A limited number of large-scale clinical studies have evaluated the association of DPP4i and the progression of retinopathy in patients with diabetes. The strength of the present study is that, to the best of our knowledge, it is the first observational investigation of the association of an add-on DPP4i in DR progression in a population-based cohort. By systemically assessing the possible confounding factors and making adjustments through PSM, we minimized detection bias and balanced the clinical characteristics between the groups. The approximately 2.5-year follow-up also means that the present findings demonstrate the long-term impacts—as opposed to the short-term effects—of DPP4i use. The potential harm that may accompany DPP4i use indicated in the present study raises substantial concerns regarding its safe use as an antidiabetic.

This study has some limitations. First, because only patients older than 40 years were included, the present findings cannot be extrapolated to other age groups. Moreover, because the patients were all Taiwanese, it remains unclear whether our findings are generalizable to other populations. Second, we could not completely prevent confounding effects. Nevertheless, we performed matching by systematically considering various variables, minimizing any imbalance between the groups. Third, we could not obtain information on the patients' diabetes control, as well as the hypoglycemic events, which are important factors of diabetes management. Nevertheless, we have matched the patients in the two groups based on their hypoglycemic agent use. Fourth, data on laboratory tests, such as the serum glucose level or hemoglobin A1c, are not available in the NHIRD. Rapid reduction of hemoglobin A1c may affect early worsening of DR [8]. However, this phenomenon should be counterbalanced in a long-term observation in the patients with better glucose control, which has been reported in the Semaglutide Unabated Sustainability in Treatment of Type 2 Diabetes (SUSTAIN) study [38]. In our study, the follow-up period of 2.5 years is comparable with the previous study, and our case number (*n* = 20,444 in both study and control groups) is higher than the SUSTAIN study (*n* = 8105 across the SUSTAIN 1 to 6 studies) [38]. Whether the DR progression in the add-on DPP4i use is related to

rapid hypoglycemic response needs further study. Fifth, the between-patient variation in diabetes severity (with some patients in severe condition) means that the alleviation of systemic disorders with medications remains challenging. The blood pressure change in our study was also not available. Nevertheless, we have matched the groups according to their disease duration, complications, medications, and underlying conditions. Therefore, the clinical characteristics of patients in the two groups were comparable at least in theory. Last, the database did not contain the results of ocular exams including optical coherence tomography, which is essential to differentiate the involvement of diabetic macular edema. The association of DPP4i and the involvement of diabetic macular edema may need further investigations. A prospective randomized trial may be required for understanding the possible effect of add-on DPP4i therapy in the progression of DR in patients with type 2 diabetes.

### **5. Conclusions**

In conclusion, add-on DPP4i therapy may be associated with the progression of preexisting DR in patients with type 2 diabetes aged ≥40 years, but the cause and effect need further research DPP4i therapy did not increase the risk of cardiovascular events. Therefore, when choosing hypoglycemic treatments for patients with diabetes and preexisting DR, the possible promoting effect of DPP4i on DR progression should be considered. A close retinal evaluation may be necessary for long-term DPP4i administration.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/jcm10132871/s1, Table S1: ICD-9 CM diagnostic codes used in the study.

**Author Contributions:** Y.-S.H. and T.-H.C. have full access to the data and takes overall responsibility. Conception and design: E.Y.-C.K., T.-H.C. and Y.-S.H.; Data collection and collation: C.K., W.-C.W. and C.-C.S.; Data analysis and interpretation: K.-J.C. and C.-C.L.; Writing: E.Y.-C.K. and C.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was supported by the Ministry of Science and Technology, Taiwan (MOST 106-2314-B-182A-045 -MY3), and Chang Gung Memorial Hospital, Taiwan (BMRPF29). The sponsor had no role in the design or conduct of this research.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of Chang Gung Memorial Hospital, Taiwan (No. 201800199B1).

**Informed Consent Statement:** Patient consent was waived due to the de-identified database.

**Data Availability Statement:** The data used for the current study cannot be made publicly available according to the NHIRD regulations of personal data protection, allowing only the person responsible for the data management to approach the data after approval from Taiwan NHI bureau.

**Acknowledgments:** We thank two biostatisticians, Alfred Hsing-Fen Lin and Ben Yu-Lin Chou, for their valuable assistance with the statistical analysis in the present study.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

## *Review* **Pneumatosis Intestinalis Induced by Alpha-Glucosidase Inhibitors in Patients with Diabetes Mellitus**

**Blake J. McKinley 1, Mariangela Santiago 2, Christi Pak 2, Nataly Nguyen 2 and Qing Zhong 2,\***

**Abstract:** Alpha-glucosidase inhibitor (αGIs)-induced pneumatosis intestinalis (PI) has been narrated in case reports but never systematically investigated. This study aimed to investigate the concurrency of PI and αGIs. A literature search was performed in PubMed, Google Scholar, WorldCat, and the Directory of Open-Access Journals (DOAJ) by using the keywords "pneumatosis intestinalis", "alpha-glucosidase inhibitors", and "diabetes". In total, 29 cases of αGIs-induced PI in 28 articles were included. There were 11 men, 17 women, and one undefined sex, with a median age of 67. The most used αGI was voglibose (44.8%), followed by acarbose (41.4%) and miglitol (6.8%). Nine (31%) patients reported concomitant use of prednisone/prednisolone with or without immunosuppressants. The main symptoms were abdominal pain (54.5%) and distention (50%). The ascending colon (55.2%) and the ileum (34.5%) were the most affected. Nineteen (65.5%) patients had comorbidities. Patients with comorbidities had higher rates of air in body cavities, the portal vein, extraintestinal tissues, and the wall of the small intestine. Only one patient was found to have non-occlusive mesenteric ischemia. Twenty-five patients were treated with conservative therapy alone, and two patients received surgical intervention. All patients recovered. In conclusion, comorbidities, glucocorticoids, and immunosuppressants aggravate αGIs-induced PI. Conservative therapy is recommended when treating αGIs-induced PI.

**Keywords:** pneumatosis intestinalis; diabetes; alpha-glucosidase inhibitors; acarbose; voglibose; miglitol; comorbidities; concomitant drugs; prednisone; immunosuppressants

### **1. Introduction**

Pneumatosis intestinalis (PI) is a condition in which gas is present within the walls of the intestines [1]. It is characterized by gas and free air in the mucosa, submucosa, and subserosa, and it can present in linear and/or cystic forms [2]. It is also called pneumatosis cystoides intestinalis. The incidence of PI is 2/6553 (0.03%) in autopsies [3]. There are two subtypes of PI: primary/idiopathic and secondary; secondary PI consists of up to 85% of all PI cases in adults [1,2].

PI is diagnosed via imaging techniques that include X-ray, computed tomography (CT), and endoscopy [4]. CT is the most sensitive medium for diagnosing PI.

PI can cause a wide range of symptoms with varying levels of severity. Some patients are asymptomatic, while others have life-threatening symptoms [5]. When patients with PI are asymptomatic, they may go undiagnosed [6]. On the other hand, the rupture of the subserosal cysts of PI could result in pneumoperitoneum without clinical peritonitis, and portal venous gas is often associated with pathological lesions [3]. Some PI cases may be secondary to transmural ischemia or necrosis of the gastrointestinal wall [7]. The mortality rates of PI increase in patients with bowel obstruction, toxic megacolon, cecal ileus, bone marrow transplants, and collagen vascular diseases [8].

Given that PI is a rare finding, its etiology is not well understood. However, PI has been reported in association with different disorders, including inflammatory bowel diseases,

**Citation:** McKinley, B.J.; Santiago, M.; Pak, C.; Nguyen, N.; Zhong, Q. Pneumatosis Intestinalis Induced by Alpha-Glucosidase Inhibitors in Patients with Diabetes Mellitus. *J. Clin. Med.* **2022**, *11*, 5918. https:// doi.org/10.3390/jcm11195918

Academic Editor: Fernando Gómez-Peralta

Received: 8 September 2022 Accepted: 2 October 2022 Published: 7 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

cytomegalovirus (CMV) colitis, acquired immunodeficiency syndrome (AIDS), emphysema, chronic obstructive pulmonary disease (COPD), cystic fibrosis, asthma, diabetes, cancer, organ transplants, fecal impaction, and mesenteric ischemia and necrosis [6,8,9].

While uncommon, some drugs are suspected of causing PI [8]. In particular, PI has been described in patients receiving corticosteroids, immunosuppressants, anticancer drugs, alpha-glucosidase inhibitors (αGIs), lactulose, and sorbitol [2,8,10]. In a multicenter study in Japan, out of 167 PI patients, 31 (19%) cases were related to diabetes. Among those 31 patients, 74.2% of them (23/31) had used αGIs [11].

αGIs competitively inhibit the intestinal α-glucosidase, thus delaying carbohydrate absorption in the small intestine. αGIs are commonly used to treat diabetes, especially type II diabetes. It has been thought that αGIs increase gastrointestinal luminal gas, contributing to the development of PI [12]. This raises a question: why does PI occur in some patients but not in others? So far, there have not yet been any systematic investigations on αGIs and PI. This study investigated the concurrency of PI and αGIs in patients with diabetes and sought to identify what other factors precipitate αGIs-induced PI.

### **2. Methods**

### *2.1. Literature Search*

A literature search was performed up to 7 June 2022, in PubMed/MEDLINE, World-Cat, Google Scholar, and the Directory of Open-Access Journals (DOAJ). We searched for articles using the following keywords: "pneumatosis intestinalis" AND "diabetes", or "pneumatosis intestinalis" AND "alpha-glucosidase inhibitors", or "acarbose" OR "voglibose" OR "miglitol" AND "pneumatosis intestinalis". Inclusion criteria were the following: clinical trials/observational studies/case series or case reports that identified patients with intramural intestinal air, the usage of alpha-glucosidase inhibitors, and studies written in English. Exclusion criteria were the following: congress abstracts, and no alpha-glucosidase inhibitors involved.

All authors participated in the initial screening. Data extraction was performed by all authors and confirmed independently by two authors (B.M. and Q.Z.) to assess for correctness and bias. Data syntheses and analyses were performed by one author (Q.Z.) and confirmed by another (M.S.). Interpretations of the results were agreed upon by all authors.

### *2.2. Statistical Analysis*

The characteristics of αGIs-induced PI cases were compared in patients with or without comorbidities, and between patients treated with voglibose and patients treated with acarbose, using the Student's t-test or X2 test with a *p* < 0.05 as statistically significant.

### **3. Results**

### *3.1. Articles Included*

Our search resulted in 151 unique titles. All titles were screened, after which 30 fulltext articles were assessed for eligibility. Ultimately, 28 articles with a total of 29 cases met our inclusion criteria, as shown in Figure 1. No clinical trials and observational studies were found. Details regarding patients' information and characters are provided in Supplemental Tables S1 and S2 [4,12–38].

**Figure 1.** Literature search flow. Legend: A total of 151 abstracts were found from four databases of Pubmed, WorldCat, Google Scholar, and DOAJ. Twenty-eight articles met the inclusion criteria.

### *3.2. Clinical Characteristics of PI*

A total of 29 patients' general information, comorbidities, past medical histories, and medication uses (Sections 3.2–3.4) are shown in Table 1. Twenty-three patients were diagnosed with type II diabetes, and six patients were diagnosed with steroid-induced diabetes as their diabetes occurred after prednisone use [23,25,26,28,34].

Eleven patients were men, 17 were women, and the sex of one patient was not defined. Ages ranged from 48 to 87, with a median of 67. The duration of time with diabetes varied from 2 days to 20 years.

Nineteen (65.5%) patients had comorbidities. Seven cases had connective tissue disorders/autoimmune diseases, including dermatomyositis [25], neuropsychiatric systemic lupus erythematosus [28], polymyalgia rheumatica [32], rheumatoid arthritis [34], granulomatosis with polyangiitis [34], hypothyroidism [22], and myasthenia gravis [26]. Three cases had immunocompromising conditions, including post-lung transplantation [38], minimal change disease-nephrotic syndrome [24], and non-specific interstitial pneumonitis (NSIP) [23]. Three patients had concomitant infections: one with acute cholecystitis [31], one with *E.coli* sepsis [24], and another with *Pseudomonas putida* detected in peritoneal dialysate effluent [37]. Five patients had hypertension, three of them with ischemic disease (post-cerebral infarction, vascular ischemia, heart ischemia, and/or nonocclusive mesenteric ischemia (NOMI)) and/or kidney failure [27,29,30,33,37].

Only 10 (34.5%) patients had no comorbidities. The number of patients with comorbidities was 1.9 times the number of patients without comorbidities.

**Table 1.** Clinical characteristics of patients with pneumatosis intestinalis.

### *3.3. Type of αGIs*

All three available αGIs—acarbose, voglibose, and miglitol—were related to PI. The most common one was voglibose (44.8%), followed by acarbose (41.4%). There were only two cases of miglitol (6.9%). Two cases did not define a specific alpha-glucosidase inhibitor [17,30].

### *3.4. Concomitant Use of Other Drugs*

As shown in Table 1, nine (31%) patients used prednisone or prednisolone ± other immunosuppressants or cytotoxic drugs [23–26,28,32,34,38]. Among those cases, one was combined with mizoribine (inhibiting guanosine synthesis), one with methotrexate, and one with tacrolimus [24,25,38]. Other antidiabetic drugs that were used consisted of insulin (seven cases), sulfonylurea (four cases), dipeptidyl peptidase-4 inhibitors (two cases), and metformin (one case). One patient used the carbohydrate supplement maltitol [22].

### *3.5. Symptoms*

Characteristics of PI, including symptoms; diagnostic imaging; complications; the segments involved; the presence of free gas in cavities or other tissues; treatment; and outcomes (Sections 3.5–3.11) are shown in Table 2.

Abbreviations: CT: computed tomography; PI: pneumatosis intestinalis.

Patients presented with symptoms in 22/29 (75.9%) of the cases. As shown in Figure 2, the most common symptoms were abdominal pain (54.5%) and abdominal distention (50%), followed by diarrhea (22.7%), bloody stool (22.7%), and constipation (13.6%).

**Figure 2.** Overview of the symptoms of αGIs-induced PI. Legend: abdominal pain and distention were the most common symptoms, followed by diarrhea and bloody stool.

### *3.6. Segment of Bowel Involved*

The location of PI had a wide distribution throughout the intestines. Nineteen (65.5%) cases had only large intestine involvement, seven (24.1%) had only small intestine involvement, and three (10.3%) had combinations of small and large intestine involvement. The ascending colon was the most involved (16/29, 55.2%), followed by the ileum (10/29, 34.5%).

### *3.7. Free Air in the Portal Vein and Intraabdominal Cavities and Extraintestinal Involvement*

As shown in Table 3, patients with comorbidities had significantly higher rates of free air in the peritoneum or retroperitoneum (9/19 (47.4%) vs. 1/10 (10%), *p* < 0.05), and with small intestine involvement (8/19 (42.1%) vs. 2/10 (20%), *p* < 0.05), compared to patients without comorbidities. Only in patients with comorbidities was free air found in the mediastinum, pericardium, or subcutaneous space (2/19 (10.5%) vs. 0/10 (0%)) or the portal vein (3/19 (15.8%) vs. 0/10 (0%)).

**Table 3.** Comparison between patients with and without comorbidities.

\* *p* < 0.05 compared to patients without comorbidities.

### *3.8. Comparison between Patients Treated with Acarbose and Patients Treated with Voglibose*

As shown in Table 1, the duration range for voglibose usage was 2 days to 10 years, with 6/13 (46.2%) having a duration shorter than 2 months. The duration range for acarbose usage was 1 year to 12 years, with 5/12 (41.7%) having a duration of less than five years. The median time of voglibose usage was relatively shorter than that of acarbose usage (0.6 years vs. 5 years).

As shown in Table 4, 7/12 (58.3%) patients who used acarbose and 10/13 (76.9%) patients who used voglibose had comorbidities. The durations of voglibose usage were shorter than those of acarbose regardless of the presence of comorbidities (without comorbidities: median 0.17 years vs. 3 years; with comorbidities: median 1.7 years vs. 8 years).

**Table 4.** Comparison between patients who used acarbose and patients who used voglibose.

\*\* *p* < 0.01 compared to the acarbose group with comorbidities.

The voglibose group with comorbidities had a significantly higher ratio of concomitant usage of glucocorticoids compared to the acarbose group with comorbidities (80% vs. 14.3%, *p* < 0.01). The three cases of glucocorticoids + immunosuppressants were all in the voglibose group. Free air in the mediastinum, pericardium, or subcutaneous space only developed in the voglibose group with comorbidities (two cases, 20%).

### *3.9. Diagnosis*

All cases of PI were confirmed by X-ray and computed tomography. Eleven (37.9%) patients were also checked with colonoscopy, and two patients were examined with endoscopic ultrasonography [26,35].

### *3.10. Treatment and Complications*

The αGIs were recognized as potential causal drugs in all cases and were terminated. Twenty-five (86.2%) patients were only treated with conservative therapy: fasting was initiated at the onset of the disease in 12 (41.4%) patients, fluid supplementation in eight

(27.5%) patients, antibiotic therapy in seven (24.1%) patients, and O2 therapy in six (20.6%) patients. One case delayed terminating the αGIs treatment until three months later, when the PI disappeared [35]. This same patient also received unique endoscopic needle puncture and high-frequency electro-scission of submucosal air cysts [35].

An exploratory laparotomy ruled out ischemia and necrosis in two (6.9%) patients with portal venous gas; therefore, no surgical intervention was needed, and the patients were treated with conservative therapy [31,33]. One case of portal venous gas was confirmed to have non-occlusive mesenteric ischemia by CT and endoscopic ultrasound examination, and the patient developed peritonitis and hypotension [37]. This case was treated with antibiotics, vasopressors, and continuous hemodiafiltration [37]. One patient developed *E. coli* sepsis that resulted in disseminated intravascular coagulation (DIC), acute renal failure, and acute respiratory distress syndrome [24]. This patient was treated with antibiotics, continuous hemofiltration, and mechanical ventilation [24].

Surgical intervention was performed in two cases. Laparotomy and hemicolectomy were performed on one patient [17], and a sigmoidectomy was performed on another to release a sigmoid volvulus [36].

### *3.11. Outcome or Duration of PI*

All patients completely recovered. Symptom resolution ranged from 4 to 90 days, with a median of 7 days recorded in 16 (55.2%) patients. Resolution of PI was confirmed via images (CT and/or X-ray) or colonoscopy in 4 to 180 days, with a median of 18 days in 22 (75.9%) patients.

Although patients with comorbidities had increased rates of free air in cavities or extraintestinal tissues compared to patients without comorbidities, the median time of disappearance of PI was similar between those two groups, with a median of 21 to 21.5 days, as shown in Table 3.

### **4. Discussion**

### *4.1. Mechanisms of αGIs-Induced PI*

We summarized 29 cases of αGIs-induced PI. The number of patients with other comorbidities was 1.9 times the number of patients without comorbidities. The exact pathophysiological mechanism of PI is unclear, but there are three theories: "mechanical theory" (the air penetrates the bowel wall), "bacterial theory" (gas-forming bacteria penetrate the submucosa), and "biochemical theory" (luminal carbohydrates are fermented, increasing intraluminal pressure) [2,36]. Based on those theories, the authors propose that multiple factors contribute to the development of αGIs-induced PI: increased production of intestinal gas, the hypomotility of the gastrointestinal tract, weakened intestinal mucosa and wall, and/or air carried from the lungs. These mechanisms are depicted in Figure 3.

Increased production of intestinal gas could be due to αGIs, supplemental carbohydrates, and bacterial infection. αGIs lead to an increase in intestinal gas by suppressing carbohydrate absorption, resulting in the retention of carbohydrates in the lumen of the intestine. The unabsorbed carbohydrates are subsequently fermented by normal flora to produce carbon dioxide, methane, and hydrogen [22,35]. An additional carbohydrate supplement maltitol, a natural sweetener that is considered a sugar alcohol, or polyol may have played a concomitant role in one case of αGIs-induced PI as it is not readily absorbed in the small intestine and thus is fermented in the large intestine [22]. Three cases had bacterial infections [21,31,37]. Bacterial infection, especially gas-producing bacteria, not only increases gas production in the lumen of the intestine but also can invade and produce gas within the intestinal wall [39].

Hypomotility of the gastrointestinal (GI) tract will prolong the fermentation of carbohydrates and increase luminal pressure. In our series of cases, the hypomotility of the GI tract could be caused by diabetic autonomic nerve damage, hypothyroidism [22], dolichocolon (an abnormally long large intestine) [36], and atrophy and fibrosis of smooth-muscle

cells of GI in dermatomyositis [25]. Increased intraluminal pressure could cause mechanical damage to the intestinal mucosa, resulting in gas migration into the intestinal wall [36].

**Figure 3.** Possible mechanisms of αGIs-induced PI. Legend: There are multiple contributors to the development of αGIs-induced PI: increased production of intestinal gas, hypomotility of the gastrointestinal tract, weakened intestinal mucosa and wall, and/or air carried from the lungs. Abbreviations: IBD: inflammatory bowel disease; GI: the gastrointestinal tract.

The intestinal mucosa and wall could be weakened or damaged by inflammation, infection, or low blood perfusion. In our case series, there were two cases of colitis [4,35], three cases of infection [24,31,37], five cases of hypertension with or without vasculopathy (one of them with non-occlusive mesenteric ischemia), five cases of connective tissue disorders [25,28,32,34], and three cases of autoimmune diseases [22,23,26]. Bowel inflammatory diseases, including ulcerative colitis and Crohn's disease, have been reported to be related to PI [9]. Hypertension and vasculopathy are important contributors to mesenteric ischemia in the elderly [16]. Connective tissue disorders could cause vasculitis or atrophy and fibrosis of smooth muscles of the GI tract, resulting in hypomotility [40].

Some drugs could also weaken or damage the intestinal mucosa and wall as well. In our series of cases, nine patients used prednisone/prednisolone, with three of them adding immunosuppressants or cytotoxic drugs [23–26,28,32,34,38]. Prednisone and immunosuppressants could deplete lymphocytes and shrink Peyer's patches, facilitating gas entry into the intestinal wall [41]. Mizoribine and methotrexate are cytotoxic and may cause mucosa and epithelial cell damage directly [2]. In our case series, the patient with dermatomyositis treated with prednisolone and methotrexate was the only one developing subcutaneous air in the cervical area, along with pneumomediastinum, pneumoperitoneum, pneumoretroperitoneum, and PI [25].

Furthermore, a pulmonary cause needs consideration. Pulmonary diseases, such as chronic obstructive pulmonary disease, asthma, cystic fibrosis, and chronic cough, may force gas to enter the blood vessels and be carried from the lungs to the intestines [8]. In Hisamoto et al.'s study, the patient had non-specific interstitial pneumonitis; after using prednisone and voglibose, the patient presented with pneumomediastinum, pneumopericardium, pneumoretroperitoneum, and PI [23]. In Otsuka et al.'s study, the patient was a lung transplantation recipient for 1031 days and had pneumonia 47 days before finding

PI [38]. In these two cases, chronic cough-induced alveolar rupture and PI could not be completely ruled out.

Patients with comorbidities not only have a higher risk of developing αGIs-induced PI but also have more severe imaging findings. Patients with comorbidities had a higher incidence of free air in cavities or extraintestinal tissues and a higher rate of small intestine involvement. Portal venous gas was only found in three patients, all of whom had comorbidities [31,33,37]. Only one of the three cases was confirmed to have non-occlusive mesenteric ischemia [37]. Although portal venous gas may suggest intestinal ischemia [42], it has been reported that 30% of patients with portal venous gas and PI were due to benign idiopathic causes [43].

### *4.2. Comparison of Three αGIs*

αGIs are especially effective in reducing postprandial hyperglycemia and are frequently used to treat patients with type II diabetes in combination with other antidiabetic drugs. The αGIs' effect on lowering blood glucose is modest, but αGIs have the advantage of a very low risk of hypoglycemia compared to sulfonylurea drugs. αGIs often cause side effects of abdominal distention, flatulence, diarrhea, and abdominal pain. Because of the side effects, αGIs should not be used in patients who have gastrointestinal disorders [44–46].

PI is a rare side effect of αGIs. Voglibose and acarbose were more commonly reported than miglitol in causing PI. This may be due to different pharmacokinetics. Acarbose and voglibose are poorly absorbed in the intestine and primarily excreted in the feces, with approximately 30% that undergo fermentation by colonic microbiota [44]. In contrast, miglitol is absorbed by the gut and excreted unchanged in the kidneys [45]. Fermentation of voglibose and acarbose may increase luminal gas.

We found that PI developed more rapidly in patients treated with voglibose than in patients treated with acarbose. In our case series, patients with comorbidities on voglibose treatment had a higher ratio of simultaneous usage of glucocorticoids/immunosuppressants than those on acarbose treatment. As we discussed in Section 4.1, concomitant glucocorticoids/immunosuppressants may precipitate the development of PI. In addition, voglibose is 190 to 270 times more potent than acarbose [46]. Therefore, the authors hypothesize that a higher ratio of concomitant usage of glucocorticoids/immunosuppressants and a higher potency of voglibose contribute to a short duration of time that is needed to trigger PI compared to that with acarbose. Clinicians should be alerted that voglibose may cause PI in the first few months of its usage, whereas acarbose has the propensity to cause PI at any time during its usage, even a decade after its initiation.

### *4.3. Symptoms and Treatments*

The most common symptoms are abdominal distension and pain, followed by diarrhea and bloody stool in the cases we analyzed. As these are non-specific symptoms, physicians should have a high level of suspicion for possible PI in patients that present with gastrointestinal complaints while taking αGIs. Additionally, physicians should be aware that PI may be identified incidentally on radiological imaging, endoscopy, or laparotomy in patients taking αGIs because several of the reviewed cases were asymptomatic.

The majority of patients fully recovered after conservative therapy, which includes the termination of alpha-glucosidases, fasting, fluid supplementation, antibiotics, and inhalation of oxygen. The authors recommend conservative treatment to be the mainstay of treatment in αGIs-induced PI.

We recommend sustaining from surgery, including exploratory laparotomies, if patients are stable without guarding or rebound tenderness. Taking into context the clinical presentation of the patients, PI and pneumoperitoneum or portal venous gas should not be used as an indication by themselves for an exploratory laparotomy. Additional criteria, including higher C-reactive protein concentrations, higher white blood cell counts, higher

lactate levels, and ascites, may be required to indicate inflammatory syndrome and the likelihood of intestinal necrosis from mesenteric ischemia [7,47,48].

It is worth mentioning that although our case series had mild clinical features in most cases with patients recovering, severe complications from PI such as perforation and death have been reported in PI patients of other etiologies [2,7,47]. It is important to combine detailed history, laboratory, and image examinations to make differential diagnoses. While it is beneficial to avoid unnecessary laparotomy in patients with non-inflammatory signs, it is also important to keep patients whose clinical features worsen under careful observation.

### *4.4. Strength and Limitations*

The strength of this study is its analysis of the contributions to PI from three alphaglucosidase inhibitors, comorbidities, and other offending drugs. To our knowledge, this is the first thorough review of αGIs-induced PI.

This review, however, also has some limitations. Due to the low incidence rate, we cannot calculate the actual prevalence of αGIs-induced PI. Moreover, this study is only a description and observation, and no correlation or causative assessment has been generated. Furthermore, the possibilities of PI induced by other types of antidiabetic drugs have not been analyzed.

### **5. Conclusions**

Alpha-glucosidase inhibitors are related to the development of PI. The most common ones are voglibose and acarbose. Voglibose usage may cause a much more rapid development of PI than acarbose. Patients with comorbidities and concurrent usage of glucocorticoids and/or immunosuppressants have a relatively higher risk of developing αGIs-induced PI with complications of free gas in cavities, portal veins, and extraintestinal tissues. The authors propose that multiple factors contribute to the development of PI when using αGIs. Intestinal ischemia or necrosis in αGIs-induced PI is uncommon, with only 1/29 (3.4%) patients having non-occlusive mesenteric ischemia in our study. The majority of patients recovered after conservative therapy. Therefore, the authors advocate for conservative therapy and the avoidance of any unnecessary surgery.

**Supplementary Materials:** The following supporting information can be downloaded at https://www. mdpi.com/article/10.3390/jcm11195918/s1, Table S1: Clinical features of patients without comorbidities (*n* = 10) [12–21], and Table S2: Clinical features of patients with comorbidities (*n* = 19) [4,22–38] can be found in the supplementary materials.

**Author Contributions:** B.J.M. and Q.Z. contributed to the conception and design of the study; all authors contributed to screening and sorting the literature; Q.Z. and M.S. analyzed the data; all authors contributed to the first draft of the manuscript; M.S., C.P. and N.N. edited the manuscript; B.J.M. critically revised the manuscript; and Q.Z. finalized the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** All authors declare no conflict of interest.

### **References**

## *Article* **The Sodium-Glucose Co-Transporter 2 (SGLT2) Inhibitor Empagliflozin Reverses Hyperglycemia-Induced Monocyte and Endothelial Dysfunction Primarily through Glucose Transport-Independent but Redox-Dependent Mechanisms**

**Dilvin Semo 1, Julius Obergassel 1, Marc Dorenkamp 1, Pia Hemling 1, Jasmin Strutz 2, Ursula Hiden 2, Nicolle Müller 3, Ulrich Alfons Müller 3, Sajan Ahmad Zulfikar 4, Rinesh Godfrey 1,5,\*,† and Johannes Waltenberger 5,6,7,\*,†**

**Abstract:** Purpose: Hyperglycaemia-induced oxidative stress and inflammation contribute to vascular cell dysfunction and subsequent cardiovascular events in T2DM. Selective sodium-glucose co-transporter-2 (SGLT-2) inhibitor empagliflozin significantly improves cardiovascular mortality in T2DM patients (EMPA-REG trial). Since SGLT-2 is known to be expressed on cells other than the kidney cells, we investigated the potential ability of empagliflozin to regulate glucose transport and alleviate hyperglycaemia-induced dysfunction of these cells. Methods: Primary human monocytes were isolated from the peripheral blood of T2DM patients and healthy individuals. Primary human umbilical vein endothelial cells (HUVECs) and primary human coronary artery endothelial cells (HCAECs), and fetoplacental endothelial cells (HPECs) were used as the EC model cells. Cells were exposed to hyperglycaemic conditions in vitro in 40 ng/mL or 100 ng/mL empagliflozin. The expression levels of the relevant molecules were analysed by RT-qPCR and confirmed by FACS. Glucose uptake assays were carried out with a fluorescent derivative of glucose, 2-NBDG. Reactive oxygen species (ROS) accumulation was measured using the H2DFFDA method. Monocyte and endothelial cell chemotaxis were measured using modified Boyden chamber assays. Results: Both primary human monocytes and endothelial cells express SGLT-2. Hyperglycaemic conditions did not significantly alter the SGLT-2 levels in monocytes and ECs in vitro or in T2DM conditions. Glucose uptake assays carried out in the presence of GLUT inhibitors revealed that SGLT-2 inhibition very mildly, but not significantly, suppressed glucose uptake by monocytes and endothelial cells. However, we detected the significant suppression of hyperglycaemia-induced ROS accumulation in monocytes and ECs when empagliflozin was used to inhibit SGLT-2 function. Hyperglycaemic monocytes and endothelial cells readily exhibited impaired chemotaxis behaviour. The co-treatment with empagliflozin reversed the PlGF-1 resistance phenotype of hyperglycaemic monocytes. Similarly, the blunted VEGF-A responses of hyperglycaemic ECs were also restored by empagliflozin, which could be attributed to the restoration of the VEGFR-2 receptor levels on the EC surface. The induction of oxidative stress completely recapitulated most of the aberrant phenotypes exhibited by hyperglycaemic monocytes and endothelial cells, and a general antioxidant N-acetyl-L-cysteine (NAC) was able to mimic the effects of empagliflozin. Conclusions: This study provides data indicating

**Citation:** Semo, D.; Obergassel, J.; Dorenkamp, M.; Hemling, P.; Strutz, J.; Hiden, U.; Müller, N.; Müller, U.A.; Zulfikar, S.A.; Godfrey, R.; et al. The Sodium-Glucose Co-Transporter 2 (SGLT2) Inhibitor Empagliflozin Reverses Hyperglycemia-Induced Monocyte and Endothelial Dysfunction Primarily through Glucose Transport-Independent but Redox-Dependent Mechanisms. *J. Clin. Med.* **2023**, *12*, 1356. https:// doi.org/10.3390/jcm12041356

Academic Editor: Fernando Gómez-Peralta

Received: 19 January 2023 Accepted: 2 February 2023 Published: 8 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the beneficial role of empagliflozin in reversing hyperglycaemia-induced vascular cell dysfunction. Even though both monocytes and endothelial cells express functional SGLT-2, SGLT-2 is not the primary glucose transporter in these cells. Therefore, it seems likely that empagliflozin does not directly prevent hyperglycaemia-mediated enhanced glucotoxicity in these cells by inhibiting glucose uptake. We identified the reduction of oxidative stress by empagliflozin as a primary reason for the improved function of monocytes and endothelial cells in hyperglycaemic conditions. In conclusion, empagliflozin reverses vascular cell dysfunction independent of glucose transport but could partially contribute to its beneficial cardiovascular effects.

**Keywords:** diabetes mellitus; empagliflozin; SGLT-2; monocytes; endothelial cells; vascular dysfunction; chemotaxis; reactive oxygen species (ROS); glucose transport; VEGFR-2; VEGFR-1

### **1. Introduction**

Diabetes mellitus-associated hyperglycaemia is a significant risk factor for developing cardiovascular disease (CVD) and associated cardiovascular mortality [1]. Diabetic vascular disease is responsible for a 2–4-fold rise in the development of coronary artery disease (CAD) [2,3]. Oxidative stress plays a vital role in developing complications associated with diabetes by inducing vascular cell dysfunction [4]. There are a variety of pathways through which hyperglycaemia transduces its deleterious effects downstream. The induction of inflammation through the activation of NF-κB pathway is one of the major determinants contributing to vascular complications in diabetes [5]. Prolonged hyperglycaemia-induced advanced glycation end products (AGEs) through the Receptor for Advanced Glycation End-products (RAGE)–NF-κB pathway contribute heavily to inflammation induction in T2DM [6]. RAGE has also been implicated in mediating the dysfunction of both monocytes [7] and endothelial cells [8]. There is significant cross-talk between oxidative stress induction and AGE-RAGE signalling [9,10].

The sodium-glucose co-transporter 2 (SGLT2) is a major glucose transporter accountable for the renal reabsorption of almost 90% of the glucose from the urine [11]. SGLT-2 inhibitor empagliflozin, is used to treat T2DM and heart failure. It is considered a new therapy for cardiovascular diseases as numerous clinical trials have shown favourable outcomes. These include the EMPA-REG OUTCOME (NCT01131676) [1,12], DAPA-HF (NCT03036124) [13] and the EMPORER-Reduced (NCT03057977) [14]. The EMPA-REG OUTCOME trial demonstrated the ability of empagliflozin to reduce cardiovascular events and overall mortality in T2DM patients with higher cardiovascular risk [1,12,15], and this by far outweighs the benefits of other glucose-lowering T2DM medications such as dipeptidyl peptidase 4 inhibitors or glucagon-like peptide-1 analogues.

Several animal studies have reported the pleiotropic effects of empagliflozin, and this drug is known to give protection against high glucose level-independent diseases such as atherosclerosis [16], heart failure [17,18] and myocardial infarction [19]. Therefore, it is highly likely that the cardiovascular benefits of empagliflozin are not solely through the reduction of blood glucose levels. The favourable pleiotropic effects of empagliflozin have already been described and discussed in several studies [20,21]. Empagliflozin is known to interfere with cellular redox status by attenuating ROS generation [22,23]. Previous studies from our laboratory have shown that in the T2DM environment, monocytes [7] and endothelial cells [24] are dysfunctional due to the accumulation of reactive oxygen species (ROS). Both monocytes and endothelial cells carry out vital functions in cardiovascular physiology, and their function is compromised during T2DM conditions [7,24–26]. Indeed, the reversal of endothelial dysfunction and imparting vascular protective effects by empagliflozin has been described in both T1DM [27] and T2DM [28]. However, Empagliflozin's beneficial effects on monocyte function have not been reported so far.

The increased incidence of CAD in T2DM patients has been linked to the impaired arteriogenesis and angiogenesis found in these patient groups [29–31]. Lack of proper VEGF responses contributes to endothelial and monocyte dysfunction in T2DM [24,31–33]. The inability of T2DM endothelial cells to respond to VEGF-2 activating growth factors leads to impaired VEGFR-2-dependent processes such as proliferation, migration and angiogenesis [24,31]. Abnormalities of angiogenesis induced by T2DM directly contribute in the pathogenesis of diabetes complications [34]. Furthermore, T2DM individuals have reduced coronary collateral formation compared to non-diabetics [35]. The defective arteriogenesis is hypothesised to be due to the dysfunction of "arteriogenic" cells, leading to the disability of these cells to home to the sites of vessel growth. Monocytes from T2DM patients were found to be defective in their migratory potential towards VEGFA and PlGF-1, previously described as "VEGF resistance" [7,26,33]. VEGF resistance is based on the non-specific activation of downstream signalling pathways in vascular cells. Pre-activation results in these cells' resistance to respond to more specific signals. As the VEGFA or PlGF-1 responses are very specific for endothelial cells and monocytes, resistance to these growth factors results in monocyte and endothelial dysfunction [36,37].

Considering the importance of oxidative stress in contributing to both monocyte and endothelial dysfunction, the proposed role of empagliflozin as a redox modulator and the ability of empagliflozin to improve cardiovascular outcomes, we hypothesised that empagliflozin could circumvent both monocyte and endothelial dysfunction through a glucose transport-dependent or independent mechanism, thereby improving vascular health. Such a possibility was investigated in this study.

### **2. Results**

### *2.1. Primary Human Monocytes and Primary Endothelial Cells Express SGLT-2*

SGLT-2 is the glucose transporter is primarily expressed in the kidneys on the epithelial cells lining the first segment of the proximal tubule. Since the reports about the expression of SGLT-2 in monocytes and endothelial cells were not robust, we decided to analyse the expression pattern of SGLT-2 in these two cell types. First, we used Immortalised Human Kidney Epithelial cells (IHKE1) as a positive control to detect a positive signal for SGLT-2. We used CD14++CD16− primary monocytes and THP-1 monocytic cell line and detected the mRNA levels of SGLT-2 using RT-qCR. We used two sets of primers, one designed to span the boundaries of exons 6 and 7 and another located at exon 13 of SGLT-2, as reported previously [38]. The exon 13 primers were reported robust in amplifying the SGLT-2 gene. Figure 1A,B shows that both primary monocytes and THP-1 monocytic cells express SGLT-2 mRNA. Both set of primers amplified the SGLT-2 gene. Even though only at a 50% expression level compared to the positive control, IHKE-1 cells, both monocytic cells expressed SGLT-2 transcripts. As reported, the exon 13 primer was more efficient in amplifying the SGLT-2 gene. In order to confirm the gene product, we sequenced the product, and it was confirmed to be the SGLT-2 gene (results not shown).

Furthermore, using FACS, we reliably detected the surface levels of the SGLT-2 protein. Similar to the situation in monocytic cells, three different types of endothelial cells, the Human Umbilical Vein Endothelial Cells (HUVEC), Human Coronary Artery Endothelial Cells (HCAEC) and Human fetoplacental Endothelial Cells (HPEC), showed varying degrees of SGLT-2 gene expression, with HPEC expressing the lowest levels (Figure 1C). Interestingly, we detected the SGLT-2 transcript amplification only when we used the exon 13 primers. We could not detect any SGLT-2 transcripts reliably when the exon 6/7 was used (Figure 1D). Nevertheless, using FACS, we detected SGLT-2 protein levels on the surfaces of these three endothelial cell types and the protein expression pattern matched with the transcript levels, with HPEC showing the lowest SGLT-2 expression. (Figure 1E). Taken together, these data confirm that both monocytes and endothelial cells express SGLT-2.

**Figure 1.** Monocytic and endothelial cells express SGLT-2. (**A**,**B**) CD14++CD16 monocytes isolated from healthy individuals (*n* = 5) and THP-1 monocytic cells (*n* = 5) were analysed for the transcript levels of SGLT-2 using two different primer pairs targeting the exon 13 and exon 6/7 using RT-qPCR. Immortalised Human Kidney Epithelial cells (IHKE1) were used as a positive control. rPLO was used as the house/keeping gene to normalise the gene expression. All data are means ± SEM. (**C**) CD14++CD16 monocytes isolated from healthy individuals (*n* = 4), and THP-1 monocytic cells were analysed by flow cytometry for the surface expression of SGLT-2 compared to the signal from the isotype-specific antibody. The mean fluorescence intensity (MFI) was then quantified. All data are means ± SEM. (**D**,**E**) Human Umbilical Vein Endothelial Cells (HUVEC), Human Coronary Artery Endothelial Cells (HCAEC) and Human fetoplacental Endothelial Cells (HPEC) (*n* = 5 each) were analysed for the transcript levels of SGLT-2 using two different primer pairs targeting the exon 13 and exon 6/7 using RT-qPCR. Immortalised Human Kidney Epithelial cells (IHKE1) were used as a positive control. rPLO was used as the house/keeping gene to normalise the gene expression. All data are means ± SEM. (**F**) Human Umbilical Vein Endothelial Cells (HUVEC), Human Coronary Artery Endothelial Cells (HCAEC) and Human fetoplacental Endothelial Cells (HPEC) (*n* = 5 each) were analysed by flow cytometry for the surface expression of SGLT-2 compared to the signal from the isotype-specific antibody. The mean fluorescence intensity (MFI) was then quantified. All data are means ± SEM.

### *2.2. CD14++CD16*− *Monocytes Exposed to Hyperglycemic Conditions Do Not Exhibit an Enhanced Transmigration Phenotype*

Since there are reports that diabetic conditions upregulate the expression of SGLT-2 [39], we wondered whether hyperglycaemic conditions or diabetes could modulate the expression of SGLT-2 in monocytes and endothelial cells. For that, we used monocytic cells and endothelial cells cultured under hyperglycaemic conditions in vitro. In addition, we also used CD14++CD16− monocytes isolated from T2DM patients and human fetoplacental endothelial cells (HPEC) isolated from gestational diabetes patients. As shown in Figure 2A, hyperglycaemic conditions did not alter the expression of SGLT-2 transcripts in both THP-1

monocytic cells and primary monocytes. Furthermore, T2DM monocytes did not reveal any significant modulation of SGLT-2 expression (Figure 2B).

**Figure 2.** Diabetic conditions do not alter the expression of SGLT-2 in monocytes and endothelial cells. (**A**) CD14++CD16− monocytes isolated from healthy individuals (*n* = 4) and THP-1 monocytic cells (*n* = 4) were exposed to either normoglycemic or hyperglycaemic conditions for 48 h. The cells were then analysed for the transcript levels of SGLT-2 using primer pairs targeting the exon 13 using RT-qPCR. rPLO was used as the house/keeping gene to normalise the gene expression. All data are means ± SEM. (**B**) CD14++CD16 monocytes isolated from T2DM patients (*n* = 12) and non-T2DM individuals (*n* = 12) were analysed for the transcript levels of SGLT-2 using primer pairs targeting the exon 13 using RT-qPCR. rPLO was used as the house/keeping gene to normalise the gene expression. (**C**) Human Umbilical Vein Endothelial Cells (HUVEC), Human Coronary Artery Endothelial Cells (HCAEC) and Human fetoplacental Endothelial Cells (HPEC) (*n* = 4 each) were exposed to either normoglycemic or hyperglycaemic conditions for 48 h. The cells were then analysed for the transcript levels of SGLT-2 using primer pairs targeting the exon 13 using RT-qPCR. rPLO was used as the house/keeping gene to normalise the gene expression. All data are means ± SEM. (**D**) HPECs (Human fetoplacental Endothelial Cells) isolated from gestational diabetes patients (*n* = 6) and non-diabetic individuals (*n* = 6) were analysed for the transcript levels of SGLT-2 using primer pairs targeting the exon 13 using RT-qPCR. rPLO was used as the house/keeping gene to normalise the gene expression. (**E**) HCAECs (Human Coronary Artery Endothelial Cells) isolated from T2DM patients (*n* = 3) and non-T2DM individuals (*n* = 3) were analysed for the transcript levels of SGLT-2 using primer pairs targeting the exon 13 using RT-qPCR. rPLO was used as the house/keeping gene to normalise the gene expression. All data are means ± SEM. ns = non-significant.

Similarly, the in vitro hyperglycaemia treatment of HUVEC, HCAEC and HPEC did not alter the levels of SGLT-2 (Figure 2C). Again, neither gestational DM nor T2DM conditions were found to alter the expression levels of SGLT-2 (Figure 2D,E). These data indicate that diabetic conditions do not alter the SGLT-2 expression levels in monocytes and endothelial cells.

### *2.3. SGLT-2 Is Weakly Involved in the Glucose Transport in Both Monocytes and Endothelial Cells*

Since we detected the transcripts and surface expression of SGLT-2 in monocytes and endothelial cells, we wondered about the potential function of these transporters in these cells. In order to understand the role of SGLT-2 as a glucose transporter, we used fluorescent-tagged glucose derivative (2-NBDG) and carried out glucose transport assays. Since GLUT-dependent transport has been reported in monocytes [40] and endothelial cells [41], we employed a GLUT1 inhibitor to understand GLUT-dependent glucose transport. As shown in Figure 3A, inhibition of GLUT resulted in the significant reduction of 2-NBDG accumulation in primary monocytes. However, the inhibition of SGLT-2 using empagliflozin resulted in a steady but not significant reduction in the 2-NBDG uptake. Both GLUT and SGLT-2 inhibitors did not synergistically influence the glucose uptake (Figure 3A). A similar trend was observed for GLUT inhibition in HUVEC, but the inhibition of SGLT-2 resulted in a meagre but significant difference in the 2-NBDG accumulation (Figure 3B). For HCAEC, SGLT-2 inhibition did not result in a significant difference in the glucose transport but showed a clear tendency in that direction (Figure 3C).

**Figure 3.** SGLT-2-dependent glucose transport in monocytes and endothelial cells. (**A**) CD14++CD16− monocytes were starved without glucose and serum for 2 h, along with either GLUT inhibitor or SGLT-2 inhibitor. Afterwards, the cells were exposed to the fluorescent-tagged derivative of glucose (2-NBDG) for 2–4 h in the presence of GLUT or SGLT-2 inhibitor or both in combination. Cells were then washed and analysed for intracellular fluorescence using FACS or fluorescence plate reader. *n* = 5. All data are means ± SEM. (**B**) HUVECs were starved without glucose and serum for 2 h, along with either GLUT inhibitor or SGLT-2 inhibitor. After that, the cells were exposed to the fluorescent-tagged derivative of glucose (2-NBDG) for 2–4 h in the presence of a GLUT or SGLT-2 inhibitor or both in combination. Cells were then washed and analysed for intracellular fluorescence using FACS or fluorescence plate reader. *n* = 5. All data are means ± SEM. (**C**) HCAECs were starved without glucose and serum for 2 h, along with either GLUT inhibitor or SGLT-2 inhibitor. Afterwards, the cells were exposed to the fluorescent-tagged derivative of glucose (2-NBDG) for 2–4 h in the presence of GLUT or SGLT-2 inhibitor or both in combination. Cells were then washed and analysed for intracellular fluorescence using FACS or fluorescence spectroscopy. *n* = 5. All data are means ± SEM. ns = non-significant. \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001.

### *2.4. Hyperglycemia-Induced Monocyte Dysfunction Is Oxidative Stress-Dependent, and Empagliflozin Alleviates ROS Accumulation and Reverses Monocyte Dysfunction*

Monocytes are rendered dysfunctional in T2DM conditions. As shown in Figure 4A, hyperglycaemic monocytes cannot migrate toward a strong arteriogenic stimulus, the placental growth factor-1 (PlGF-1), which is a direct readout indicating monocyte dysfunction. This inability is partly attributed to the ligand-independent activation monocytes resulting in random motility. The random motile monocytes (termed chemokinesis) cannot sense and specifically respond to growth factor stimulation. As expected, the hyperglycaemic monocytes readily undergo chemokinesis (Figure 4B). Oxidative stress was found to be a

primary driver of monocyte dysfunction. The exogenous addition of hydrogen peroxide (H2O2) was found to be sufficient to induce monocytes' refractoriness to respond to PlGF-1 (Figure 4C). Exactly as in hyperglycaemic conditions, H2O2 treatment alone was sufficient to induce monocyte chemokinesis (Figure 4D). Since empagliflozin has several pleiotropic effects and is considered to be a redox modulator [28], we hypothesised that empagliflozin could interfere with hyperglycaemia-induced ROS accumulation. As shown in Figure 4E, empagliflozin significantly attenuated hyperglycaemia-induced ROS accumulation. Similarly, hyperglycaemia-induced monocyte dysfunction (Figure 4F) and the induction of monocyte chemokinesis (Figure 4G) were significantly reinstated by empagliflozin. Since empagliflozin does not significantly modulate glucose transport in monocytes, these beneficial effects are independent of the intracellular glucose levels.

**Figure 4.** Hyperglycaemia-induced monocyte dysfunction is reversed by empagliflozin through the modulation of oxidative stress. (**A**) CD14++CD16− monocytes were cultured in vitro in normoglycemic (5 mM glucose) or hyperglycaemic (30 mM glucose + 100 μM methylglyoxal) for 48 h and were analysed for their ability to undergo chemotaxis (directional migration) towards arteriogenic

stimuli PlGF-1. Boyden chamber assays were performed. *n* = 6. (**B**) CD14++CD16− monocytes were cultured in vitro in normoglycemic (5 mM glucose) or hyperglycaemic (30 mM glucose + 100 μM methylglyoxal) for 48 h and were analysed for their ability to undergo chemokinesis (random migration). Checkerboard analyses were performed for this. *n* = 6. All data are means ± SEM. (**C**,**D**) CD14++CD16− monocytes were cultured in vitro in normoglycemic (5 mM glucose) conditions in the presence of 200 μM H2O2 for 24 h. After that, the cells were analysed for their ability to undergo chemotaxis towards PlGF-1 and chemokinesis using Boyden chamber assays. *n* = 6. All data are means ± SEM. (**E**) CD14++CD16− monocytes were cultured in vitro in normoglycemic (5 mM glucose) or hyperglycaemic (30 mM glucose + 100 μM methylglyoxal) for 48 h. The reactive oxygen species (ROS) accumulated was detected by fluorescence spectroscopy using 5-(and-6)-carboxy-2 ,7 -difluorodihydrofluorescein diacetate (H2-DFFDA) reagent. *n* = 3. All data are means ± SEM. (**F**,**G**) CD14++CD16− monocytes were cultured in vitro under hyperglycaemic (30 mM glucose + 100 μM methylglyoxal) conditions for 48 h in the presence or absence of 100 ng/mL SGLT-2 inhibitor empagliflozin. After that, the cells were analysed for their ability to undergo chemotaxis towards PlGF-1 and chemokinesis using Boyden chamber assays. *n* = 6. All data are means ± SEM. ns = nonsignificant. \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001.

### *2.5. Oxidative Stress-Dependent Impairment of VEGFR-2 Contributes to Endothelial Dysfunction in Hyperglycemia, and Empagliflozin Restores VEGFR-2 to Alleviate Endothelial Dysfunction*

Similar to dysfunctional monocytes, diabetes conditions render endothelial cells unable to carry out their primary physiological functions. Most of the signals from the physiological functions of the endothelial cells are transduced through the Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2), and defective VEGFR-2 signalling characterises endothelial dysfunction. We tested this. The ability to respond to VEGFR-2 ligand VEGF-A was significantly reduced in hyperglycaemic endothelial cells (Figure 5A). This defect was found to be due to the reduction of VEGFR-2 surface expression (Figure 5B). Indeed, oxidative stress is vital in mediating the refractoriness of endothelial cells to VEGF-A. The treatment of endothelial cells with exogenous H2O2 readily recapitulated the impaired ability to respond to VEGF-A stimulation (Figure 5C), and the induction of oxidative stress was sufficient to impair the surface expression of VEGFR-2 levels (Figure 5D). Confirming the role of empagliflozin as a redox modulator, hyperglycaemia-induced ROS accumulated was reduced in empagliflozin-treated endothelial cells (Figure 5E). Furthermore, empagliflozin restored the ability of hyperglycaemic endothelial cells to respond to VEGF-A (Figure 5F), and this was due to the improved surface expression of the VEGFR-2 receptor (Figure 5G). Taken together, these data indicate that empagliflozin modulates redox homeostasis in endothelial cells.

### *2.6. A General Antioxidant Improves Cell Function, Whereas Induction of Oxidative Stress Reverses the Beneficial Effects of Empagliflozin*

From the results described so far, it seems likely that the modulation of oxidative stress by empagliflozin contributes to improving cell function in hyperglycaemic conditions. We used a very commonly used antioxidant, N-acetyl cysteine (NAC), in our system. In fact, NAC is known to impart a protective effect against diabetes-associated cardiovascular complications [42]. As hypothesised, the application of NAC reversed hyperglycaemiainduced monocyte and endothelial dysfunction (Figure 6A,B). NAC, such as empagliflozin, significantly restored both the impairment of PlGF-1 and VEGF-A responses. This indicated that oxidative alleviation is central to improving vascular cell function. Next, we asked if we were able to suppress the beneficial effects of empagliflozin by the induction of oxidative stress. For that, we used H2O2. Oxidative stress significantly attenuated the improvement of monocyte and endothelial cell function induced by empagliflozin in hyperglycaemic conditions (Figure 6C,D). These data further confirmed the role of empagliflozin as a redox regulator in monocytes and endothelial cells.

**Figure 5.** Empagliflozin reverses hyperglycaemia-induced endothelial dysfunction by reducing oxidative stress. (**A**) HUVECs were exposed to in vitro normoglycemic conditions (5 mM glucose) or hyperglycaemic conditions mimicking a diabetic milieu (30 mM glucose + 100 μM methylglyoxal) for 24 h. The cells were then analysed for their ability to undergo chemotaxis (directional migration) towards angiogenic stimuli VEGF-A. Boyden chamber assays were performed. *n* = 5. (**B**) HUVECs were exposed to in vitro normoglycemic conditions (5 mM glucose) or hyperglycaemic conditions mimicking a diabetic milieu (30 mM Glucose + 100 μM methylglyoxal) for 24 h. Thereafter, FACS analysis of the surface expression of VEGFR-2 on hyperglycaemic HUVECs was done. (*n* = 5). All data are means ± SEM. (**C**) HUVECs were exposed to in vitro normoglycemic conditions (5 mM glucose) in the presence of 200 μM H2O2 for 24 h. After that, the cells were analysed for their ability to undergo chemotaxis towards VEGF-A using Boyden chamber assays. *n* = 5. (**D**) HUVECs were exposed to in vitro normoglycemic conditions (5 mM glucose) in the presence of 200 μM H2O2 for 24 h. After that, the cells were analysed for the surface expression of VEGFR-2 using FACS. *n* = 4. Data are means ± SEM. (**E**) HUVECs were exposed to in vitro normoglycemic conditions (5 mM glucose) or hyperglycaemic conditions mimicking a diabetic milieu (30 mM Glucose + 100 μM methylglyoxal for 24 h. The reactive oxygen species (ROS) accumulated was detected by fluorescence spectroscopy using 5-(and-6)-carboxy-2 ,7 -difluorodihydrofluorescein diacetate (H2-DFFDA) reagent. *n* = 3. All data are means ± SEM. (**F**) HUVECs were exposed to in vitro normoglycemic conditions (5 mM glucose) or hyperglycaemic conditions mimicking a diabetic milieu (30 mM Glucose + 100 μM methylglyoxal) for 24 h in the presence or absence of 100 ng/mL SGLT-2 inhibitor empagliflozin. After that, the cells were analysed for their ability to undergo chemotaxis towards VEGF-A using Boyden chamber assays. *n* =5(**G**) FACS analysis of the surface expression of VEGFR-2 of the cells grown under conditions as described for F. *n* = 4. All data are means ± SEM. ns = non-significant. \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001.

**Figure 6.** Manipulating the redox status of the cells recapitulates the dysfunction phenotype. (**A**) CD14++CD16− monocytes were cultured in vitro in normoglycemic (5 mM glucose) or hyperglycaemic (30 mM glucose + 100 μM methylglyoxal) for 48 h in the presence or absence of 5 mM N-acetylcysteine (NAC) and were analysed for their ability to undergo chemotaxis (directional migration) towards arteriogenic stimuli PlGF-1. Boyden chamber assays were performed. *n* = 5. (**B**) HUVECs were exposed to in vitro normoglycemic conditions (5 mM glucose) or hyperglycaemic conditions mimicking a diabetic milieu (30 mM Glucose + 100 μM methylglyoxal) for 24 h in the presence or absence of 5 mM N-acetylcysteine (NAC). The cells were then analysed for their ability to undergo chemotaxis (directional migration) towards angiogenic stimuli VEGF-A. Boyden chamber assays were performed. *n* = 5. (**C**) CD14++CD16− monocytes were cultured in vitro under hyperglycaemic (30 mM glucose + 100 μM methylglyoxal) conditions for 48 h in the presence or absence of 100 ng/mL SGLT-2 inhibitor empagliflozin. In addition, cells were treated with or without 200 μM H2O2. After that, the cells were analysed for their ability to undergo chemotaxis towards PlGF-1 in the presence of 200 μM H2O2. *n* = 5. (**D**) HUVECs were exposed to in vitro normoglycemic conditions (5 mM glucose) or hyperglycaemic conditions mimicking a diabetic milieu (30 mM Glucose + 100 μM methylglyoxal) for 24 h in the presence or absence of 100 ng/mL SGLT-2 inhibitor empagliflozin. In addition, cells were treated with or without 200 μM H2O2. After that, the cells were analysed for their ability to undergo chemotaxis towards VEGF-A using Boyden chamber assays in the presence of 200 μM H2O2. *n* = 5. ns = non-significant. \*\* *p* < 0.01 and \*\*\* *p* < 0.001.

### **3. Discussion**

The present study demonstrates that the two important cell types, the monocytes and endothelial cells, are dysfunctional in hyperglycaemic conditions. These cells are required for vascular repair processes and contribute to atherosclerosis development in diabetic patients when dysfunctional. Empagliflozin, the SGLT-2 inhibitor—developed to attenuate the glucose reabsorption by the kidneys as a strategy to reduce blood glucose levels in diabetes patients—improves both hyperglycaemia-induced monocyte and endothelial cell dysfunction by glucose transport-independent mechanisms. Even though monocytes and endothelial cells express functional SGLT-2, glucose transport is not primarily mediated through SGLT-2 but via GLUT. Empagliflozin interferes with the hyperglycaemia-induced oxidative stress induction and attenuates ROS accumulation. Empagliflozin-treated hyperglycaemic monocytes displayed attenuated chemokinesis and were readily responding to arteriogenic stimuli.

Similarly, empagliflozin-treated endothelial cells displayed improved VEGFR-2 receptor levels on the cell surface and responded robustly to angiogenic stimuli. The attenuation of arteriogenesis and angiogenesis is a hallmark of diabetes mellitus contributing to microand macrovascular complications [30,34]. Other than interfering with the glucose transport in the proximal kidney tubules and contributing positively to the reduction of glucotoxicitydependent alterations in cell function, the results described here highlight the pleiotropic effects of empagliflozin and can be a contributing pathway through which this drug offers protection in diabetic and heart failure patients.

Even though SGLT-2 expression is primarily confined to the kidneys, its expression has been reported in various other cell types, including endothelial cells [23,43] and smooth muscle cells [44]. However, the expression of SGLT-2 on monocytes and monocytic cell lines was not reported. Here, we detected the mRNA and protein expression of SGLT-2 in both primary monocytes and several primary endothelial cell types. These data confirm that SGLT-2 is expressed in vascular cells. SGLT-2 expression is regulated dynamically, and several biochemical stimuli such as TNFα and Ang-II have been reported to increase its expression [23,45]. Furthermore, there are reports about the upregulation of SGLT-2 in diabetes [46,47] and heart failure patients [48]. However, our investigations using in vitro hyperglycaemic conditions and monocytes and endothelial cells from T2DM patients did not reveal any differences in the expression pattern of SGLT-2, indicating that diabetes conditions do not stimulate SGLT-2 expression in these cells. The signalling pathways responsible for the induction of SGLT2 in monocytes and endothelial cells have not been delineated.

We identified that the SGLT-2 expressed on monocytes and endothelial cells are also functionally active. Even though to a very low level, the inhibition of SGLT-2 using empagliflozin resulted in slightly altered glucose transport in both these cell types. Although the differences were minimal, there was always a tendency to downgrade glucose transport. This indicates that SGLT-2 is able to transport glucose in these cells, albeit to a lower level. These also validate our data that the positive effect of empagliflozin on monocyte and endothelial function is not secondary to the reduction of glucose transport. As expected, GLUT inhibition significantly blocked glucose transport in both monocytes and endothelial cells. This is in line with the published data on the vital role of GLUT in transporting glucose [49,50].

This study's most exciting and novel finding is the influence of empagliflozin in improving monocyte function in hyperglycaemic conditions. Even though several animal studies have shown that empagliflozin is able to improve endothelial function [27,28,51], its positive effects on monocyte function have not yet been reported. Furthermore, most of the positive effects of empagliflozin on endothelial function in vivo are also secondary to its role in reducing glucotoxicity. However, we report that empagliflozin imparts positive effects on endothelial cells and monocytes through a pleiotropic mechanism. Indeed, this ambiguous role of empagliflozin has been reported in several studies [20]. Our data refer to the specific contribution of empagliflozin in alleviating the oxidative stress-dependent induction of monocyte and endothelial cell function. Further studies are required to evaluate the improvement of endothelial function in vivo using the flow-mediated dilation (FMD) method [52].

Translational data suggest that SGLT2 inhibitors may positively affect plaque composition and burden through the reduction of inflammatory and cell adhesion pathways. Still, human data are not available to make solid conclusions. However, endothelial dysfunction represents an important mechanism underlying heart failure with preserved ejection fraction (HFpEF) [53]. Impaired coronary microvascular function is strongly associated with the severity of heart failure [54]. Even though published data do not completely support a causative role for monocytes and heart failure, their role in atrial fibrillation (AF) has been postulated [55]. Therefore, the improvement of both endothelial and monocyte function by empagliflozin could improve heart failure outcomes.

The modulation of oxidative stress by empagliflozin is a pleiotropic mechanism on which several positive effects of this drug could be based. Such a possibility is currently being tested in patients with type 2 diabetes (EMPOX study) in a clinical trial. (ClinicalTrials.gov Identifier: NCT02890745). Our data confirm the notion that empagliflozin can reduce the ROS accumulation induced by hyperglycaemia. This could be secondary to reducing oxidative stress-inducing machinery such as NADPH oxidases (NOXs) or improving the antioxidant system [56]. The dysfunction of monocytes and endothelial cells induced by hyperglycaemia is oxidative stress-dependent [24,33], and the complete alleviation of this dysfunction phenotype of empagliflozin demonstrates its function as a redox regulator. The alleviation of the beneficial effects of empagliflozin by the induction of oxidative stress demonstrates that the beneficial effects of empagliflozin are redox-dependent. Since functionally active monocytes and endothelial cells could carry out a wide array of repair and regeneration processes in diabetes, empagliflozin-mediated improvement of these two cell types would contribute to the beneficial aspects of empagliflozin. Further investigations are necessary to understand how empagliflozin is able to impart its effects as a redox modulator.

### **4. Materials and Methods**

### *4.1. Monocyte Isolation from Clinical Cohorts and Healthy Individuals*

CD14++CD16− human monocytes were isolated from healthy donors and from non-T2DM individuals or T2DM patients according to a published protocol [33] using Magnetassisted cell sorting (MACS) using negative selection with the human Monocyte Isolation Kit II from Miltenyi Biotec (Bergisch Gladbach, Germany). The study was approved by the scientific and ethics committee of the University of Münster and the University of Jena and conformed to the principles of the Declaration of Helsinki. Written informed consent was obtained from all donors by the blood bank, and thrombocyte reduction filters were provided anonymously without sharing personal and detailed information. The purity of isolated cells was confirmed by FACS, and they were around 98% pure. The clinical characteristics are described in detail in Supplementary Table S1.

### *4.2. Human Umbilical Vein Endothelial Cells Isolation and Ethics*

Human umbilical vein endothelial cells (HUVEC) were isolated from anonymously acquired umbilical cords according to the Declaration of Helsinki, "Ethical Principles for Medical Research Involving Human Subjects" (1964), as described previously [57]. The study was approved by the Jena University Hospital Ethics Committee (no. 3130-05/11), and donors were informed and gave written consent. For cell preparation, umbilical cord veins were cleaned with 0.9% NaCl solution, and cells were detached with 0.01% collagenase dissolved in M199 for 3 min at 37 ◦C. Veins were then rinsed with M199/10% FCS, and the cell suspension was centrifuged (500× *g*, 6 min). The pellet was resuspended in M199/10% FCS and seeded on a cell culture flask coated with 0.2% gelatine. After 24 h, cells were washed and cultured in full growth medium (M199, 20% FCS, 7.5 U/mL heparin, 100 U/mL penicillin and 100 μg/mL streptomycin).

### *4.3. Human Fetoplacental Endothelial Cell Isolation from Clinical Cohorts and Healthy Individuals*

Primary HPEC were isolated from arterial vessels of human term placentas obtained from healthy and GDM pregnancies, as described previously [58]. In brief, arterial vessels from the apical surface of the chorionic plate were dissected and cells were isolated by perfusion of the arteries with Hank's balanced salt solution (HBSS, Invitrogen, Waltham, MA, USA) containing 0.1 U/mL collagenase, 0.8 U/mL dispase II (Roche, Basel, Switzerland) and 10 mg/mL penicillin/streptomycin for 8 min. Digested suspension was centrifuged (200× *g*, 5 min), the cell pellet resuspended in EBM-2 Media supplemented with the EGM-2 MV Bullet Kit (Lonza, Basel, Switzerland), containing 5% human heat-inactivated serum of pregnant women instead of FCS and plated on 1% gelatine-coated wells of a 12-well plate. Cells were split into a 12 cm2 flask, 25 cm2 flask and, finally, 75 cm2 flask accordingly when cells were confluent. The identity and purity of HPEC were confirmed by immunocytochemistry staining of specific endothelial markers for von Willebrand factor and CD31 (PECAM1), fibroblast markers (CD90 and TE-7) and smooth muscle cell markers (SMA and Desmin). For maintaining a culture, primary cells were grown in EBM-2 Media supplemented with the EGM-2 MV Bullet Kit containing 5% FCS, and cells split for less than 10 passages were used for experiments. The ethics committee of the Medical University of Graz approved this study (27-265 ex 14/15). All individuals gave voluntary informed consent and underwent an oral glucose tolerance test (OGTT) at 24 weeks of gestation. Control subjects were selected based on negative OGGT. Women with GDM diagnosed according to the WHO/IADPSG criteria, but without other pregnancy complications, were recruited before delivery. All subjects included in the GDM group were managed by diet and lifestyle modifications during the remaining time of pregnancy. The study conforms to the Declaration of Helsinki. Clinical characteristics are listed in Supplementary Table S2.

### *4.4. Monocyte, HUVEC, HCAEC and HPEC Culture*

Primary human monocytes were maintained in RPMI-1640 medium (+L-glutamine, D(+)-glucose; Thermo Scientific, Waltham, MA, USA) supplemented with 5 mM glucose, 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). For migration experiments, cells were starved for 2–4 h in FBS-free RPMI-1640 medium. Monocytes were kept in an incubator at 37 ◦C and 5% CO2. Normoglycemic medium contained 5 mM glucose and 25 mM mannitol. In hyperglycaemic medium, 30 mM glucose and 100 μM methylglyoxal were used, and the cells were treated for 48 h. HUVECs were cultured in full growth medium (M199, 20% FCS, 7.5 U/mL heparin and 1% penicillin/streptomycin). In general, HUVEC from 2–5 passages were used for the experiments. For migration experiments, cells were starved for 2–4 h in FBS-free M199 medium. Monocytes were kept in an incubator at 37 ◦C and 10% CO2. Normoglycemic medium contained 5 mM glucose and 25 mM mannitol. In hyperglycaemic medium, 30 mM glucose and 100 μM methylglyoxal were used, and the cells were treated for 24 h. HCAECs were obtained from Lonza and were maintained in EBMTM-2 Basal Medium (CC-3156) and EGMTM-2 MV Microvascular Endothelial Cell Growth Medium SingleQuotsTM supplements (CC-4147), as per the recommendations of the manufacturer. Cells up to a passage of 6 were used for the experiments. HPECs were grown in EBM-2 media supplemented with the EGM-2 MV Bullet Kit containing 5% FCS, and cells split for less than 10 passages were used for experiments.

### *4.5. Reagents*

Cell culture media RPMI 1640 Medium GlutaMAXTM was obtained from Life Technologies. Human VEGF-A and PlGF-1 were from Peprotech. H2DFFDA and CellROX were from Life Technologies. Hydrogen peroxide, methylglyoxal, NAC and 2-NBDG were from Sigma Aldrich. All the primers for qPCR were custom synthesised from Sigma Aldrich. Primer sequences are described in Supplementary Table S3.

### *4.6. RNA Isolation and qPCR*

For the extraction of RNA, roughly 5–8 × 106 monocytes were used. For in vitro experiments, the RNA was extracted between 8 and 12 h post-cell treatment. Total RNA purification was performed using a NucleoSpin RNA isolation kit (Macherey-Nagel, Dueren, Germany), and cDNA was synthesised using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). qPCR was carried out using iTaq™ Universal SYBR®® Green supermix (Bio-Rad, Hercules, CA, USA) in the Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The threshold cycle (Cq) value of each sample was calculated, and the expression of the target gene mRNA relative to rplO was determined by the 2−ΔΔCt method. The sequences of the primers used can be found in Supplementary Table S3.

### *4.7. Glucose Uptake Assay*

Cells were equilibrated in glucose-free medium with 1% serum for three hours prior to being treated with 10 μM of 2-NBDG for 30 min at 37 ◦C, together with GLUT inhibitor or empagliflozin. Cells were washed two times, and the flow cytometry analysis was carried out using Guava easyCyte (Millipore, Burlington, MA, USA).

### *4.8. Monocyte Chemotaxis and Chemokinesis Assay*

Chemotaxis assays were performed as described previously [7,59] using a 48-well Boyden chamber (Neuroprobe, Gaithersburg, MD, USA) and Nucleopore PET membrane (Whatman, Maidstone, UK) with 5 μm diameter pores. Cells in a concentration from 0.5 × 106 cells/mL were allowed to migrate for 90 min at 37 ◦C and 5% CO2. The cells that migrated through the pores were counted. For quantification, migrated cells were counted by 20 high-power fields in four wells using the Axioskop 2 Plus microscope (Carl Zeiss, Jena, Germany).

### *4.9. Chemotaxis Assay of Endothelial Cells*

For the detection of endothelial cell chemotaxis, a modified 48-well Boyden chamber (Nucleopore) and a polycarbonate membrane with a pore diameter of 8 mm (Nucleopore) were used as described earlier [24,33]. Endothelial cells were cultured for 24 h under normal and high glucose conditions with or without empagliflozin. For the assay, cells were starved in a serum-free M199 medium for 1 h, trypsinised, washed and resuspended in serum-free medium. Cells were seeded in a concentration of 0.35 × 106 cells/mL and allowed to migrate for 1.5 h at 37 ◦C and 5% CO2 with and without 25 ng/mL VEGF-A stimulation. Migrated cells were fixed with 99% ethanol for 10 min and stained with Giemsa staining solution. Cells at the upper side of the filter membrane were scraped off. Migrated cells were counted using ZEISS Axioskop 2 Plus at 10X magnification from 3 different wells.

### *4.10. Intracellular Reactive Oxygen Species Detection*

HUVECs were seeded in a 12-well plate in different glucose conditions with or without 100 ng/mL empagliflozin for 24–48 h. The method was based on the modification of the published protocol [60]. After that, cells were washed twice with Krebs-Ringer phosphate glucose buffer (KRPG; 145 mM NaCl, 5.7 mM KH2PO4, 4.86 mM KCl, 0.54 mM CaCl2, 1.22 mM MgSO4 and 5.5 mM glucose) and then resuspended in 1 mL KRPG. Carboxy-H2DFFDA (20 μM) or CellROX (5 μM) was added; the suspension mixed well and then incubated in dark for 20 to 30 min at room temperature. The subsequent steps were strictly carried out in the dark. The cells were washed twice with KRPG. The fluorescence intensity was quantified with a fluorescence multimode microplate reader (Vector, Perkin Elmer) with excitation at 485 nm and emission at 530 nm or by Guava easyCyte FACS using the FITC-channel (Millipore, Burlington, MA, USA).

### *4.11. Detection of the Surface Expression of VEGFR-2*

HUVECs were cultured in normoglycemic or hyperglycaemic conditions with or without 100 ng/mL empagliflozin for 24 h in a 12-well plate. For the assay, the cells were trysinised, washed once with 1X PBS and resuspended in 500 μL of PBS/BSA (0.5% BSA) solution. Fc-R blocking reagent was added for 10 min at room temperature, and 2 μL of PE-VEGFR2 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) were added and incubated for 15 min at room temperature in the dark, and the FACS analysis was done using Guava easyCyte (Millipore, Burlington, MA, USA).

### *4.12. Statistical Analysis*

To analyse the significance of differences in experiments with monocytes isolated from diabetic or healthy individuals/mice, the Mann–Whitney Rank Sum Test (for intergroup comparisons) or Kruskal–Wallis One-Way Analysis of Variance on Ranks with Tukey's or Dunn's post hoc correction was used. For all the other experiments, two-sample independent *t*-tests or when multiple comparisons were made, Kruskal–Wallis One-Way Analysis of Variance on Ranks with Tukey's or Dunn's post hoc correction was performed. SigmaPlot 12 software was used for the statistical analysis. The level of significance was defined as *p* < 0.05. All other statistics and graphs were generated using GraphPad Prism 8 software.

### **5. Conclusions**

In conclusion, using the cell culture model of in vitro hyperglycaemia and T2DM monocytes and T2DM endothelial cells and endothelial cells from gestational diabetes patients, we identified that both monocytes and endothelial cells express SGLT-2 transcripts and harbour functionally active SGLT-2 on their surface. SGLT-2 was not responsible for the glucose transport in these cells. However, the empagliflozin treatment significantly reversed the monocyte and endothelial cell dysfunction induced by hyperglycaemia. This was completely independent of glucose transport but through the reduction of oxidative stress. Mechanistically, empagliflozin attenuated the oxidative stress-dependent chemokinesis of monocytes and restored the surface levels of VEGFR-2 on endothelial cells. Furthermore, our results highlight the pleiotropic role of empagliflozin as a redox modulator.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jcm12041356/s1: Table S1. Clinical characteristics of the non-T2DM and T2DM individuals used in the study. Table S2. Clinical characteristics of non-diabetic and diabetic individuals used for the HPEC isolation. Table S3. RT-qPCR primer sequences used in the study.

**Author Contributions:** R.G. and J.W. conceived and developed the concept; R.G., D.S., J.O., M.D. and J.W. designed the experiments and interpreted the data; D.S., J.O., M.D., P.H. and R.G. did the experiments; J.S. and U.H. isolated, characterized, provided, and supported the experiments using HPECs. N.M., U.A.M. and R.G. were involved with patient characterization, recruitment, and isolation of diabetic and non-diabetic monocytes; R.G. drafted the manuscript; S.A.Z. and J.W. contributed to the redrafting of the manuscript; R.G. and J.W. finalised the manuscript with input from all the authors and R.G. and J.W. supervised the study. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported, in part, by the Innovative Medizinische Forschung (IMF) grant GO121222 (to Godfrey), Interdisziplinäre Zentrum für Klinische Forschung (IZKF) grant IZKF-SEED 014/20 (to Dorenkamp), grants from the Deutsche Forschungsgemeinschaft (DFG), Collaborative Research Centre 656 Münster (project C12) and grants from the Deanery of the Medical Faculty of the Westfälische Wilhelms-Universität Münster (all to Waltenberger).

**Institutional Review Board Statement:** Human blood leukocyte reduction filters from healthy subjects were received from the blood bank of the University Hospital Münster. Written informed consent was obtained from all donors by the blood bank, and leukocyte reduction filters were provided anonymously without sharing personal and detailed information. This study was approved by the local ethics committee of Münster University Hospital, Germany, and Jena University Hospital, Germany. All the DM patients and non-DM individuals provided written informed consent to participate in the study. The ethical permission number is 2011-612-f-S (Münster) and 4125-06/14 (Jena). The study was conducted according to the principles of the Declaration of Helsinki.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We thank Merle Leffers and Sybille Koch for their technical support.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

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## *Article* **Sex Differences in Cardiovascular Prevention in Type 2: Diabetes in a Real-World Practice Database**

**Anna Ramírez-Morros 1,2, Josep Franch-Nadal 3,4, Jordi Real 3,4, Mònica Gratacòs 3 and Didac Mauricio 3,4,5,6,\***

**Abstract:** Women with type 2 diabetes mellitus (T2DM) have a 40% excess risk of cardiovascular diseases (CVD) compared to men due to the interaction between sex and gender factors in the development, risk, and outcomes of the disease. Our aim was to assess differences between women and men with T2DM in the management and degree of control of cardiovascular risk factors (CVRF). This was a matched cross-sectional study including 140,906 T2DM subjects without previous CVD and 39,186 T2DM subjects with prior CVD obtained from the System for the Development of Research in Primary Care (SIDIAP) database. The absolute and relative differences between means or proportions were calculated to assess sex differences. T2DM women without previous CVD showed higher levels of total cholesterol (12.13 mg/dL (0.31 mmol/L); 95% CI = 11.9–12.4) and low-density lipoprotein cholesterol (LDL-c; 5.50 mg/dL (0.14 mmol/L); 95% CI = 5.3–5.7) than men. The recommended LDL-c target was less frequently achieved by women as it was the simultaneous control of different CVRF. In secondary prevention, women showed higher levels of total cholesterol (16.89 mg/dL (0.44 mmol/L); 95% CI = 16.5–17.3), higher levels of LDL-c (8.42 mg/dL (0.22 mmol/L); 95% CI = 8.1–8.8), and higher levels of triglycerides (11.34 mg/dL (0.13 mmol/L); 95% CI = 10.3–12.4) despite similar rates of statin prescription. Recommended targets were less often achieved by women, especially LDL-c < 100 mg/dL (2.59 mmol/L). The composite control was 22% less frequent in women than men. In conclusion, there were substantial sex differences in CVRF management of people with diabetes, with women less likely than men to be on LDL-c target, mainly those in secondary prevention. This could be related to the treatment gap between genders.

**Keywords:** risk factors; cardiovascular diseases; diabetes mellitus; type 2; gender

### **1. Introduction**

According to the International Diabetes Federation (IDF), the global age-standardized prevalence of diabetes in subjects 20–79 years in 2019 was similar between men and women (9.6% and 9%, respectively) [1]. However, there were more diabetes-associated deaths among women than in men (2.3 vs. 1.9 million) [1].

Large-scale meta-analyses have consistently shown that type 2 diabetes (T2DM) confers a greater excess risk of macrovascular complications in women compared with men. The relative risk of coronary heart disease (CHD) is estimated to be 44% higher in women; the risk of stroke is 27% higher, the occlusive vascular mortality rate is nearly 50% higher,

**Citation:** Ramírez-Morros, A.;

Franch-Nadal, J.; Real, J.; Gratacòs, M.; Mauricio, D. Sex Differences in Cardiovascular Prevention in Type 2: Diabetes in a Real-World Practice Database. *J. Clin. Med.* **2022**, *11*, 2196. https://doi.org/10.3390/ jcm11082196

Academic Editor: Fernando Gómez-Peralta

Received: 7 March 2022 Accepted: 12 April 2022 Published: 14 April 2022 Corrected: 2 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and the risk of vascular dementia is 19% higher [2–4]. Regarding microvascular complications, it has been reported that the risk of end-stage renal disease is 38% higher in women than in men [2–4]. These disparities have been attributed to the interaction between sex and gender factors in the development, risk, and outcomes of diabetes [3].  Gender differences arise from inequalities in sociocultural processes (e.g., environmental influences, nutritional patterns, lifestyle, or attitudes toward treatment and prevention) [3].

The mechanisms underpinning the biological disparities in the likelihood of developing diabetes-related vascular complications between sexes are not entirely understood. Women develop diabetes at a higher body mass index (BMI) than men, and one of the proposed explanations is that they usually have lower visceral and ectopic fat, which may lead to a slower transition to insulin resistance and diabetes. As a result, women might be exposed longer to hyperglycemia or a suboptimal glucose level state, resulting in greater vascular damage and deterioration of the cardiovascular risk factors (CVRFs) [2–4]. In addition to these sex-specific differences, gender dissimilarities in diabetes management and healthcare provision may partially contribute to the diabetes-related increased CVD risk. For instance, although the recommendations on prevention, management, and treatment of diabetes and diabetes-related complications are similar for both sexes, women are less likely than men to receive guideline-recommended care [4]. Indeed, some studies have reported that women are less likely than men to be monitored for foot and eye complications, and they receive less effective management and screening of CV risk factors such as blood pressure (BP), BMI, or smoking status [2,5]. Additionally, the odds of receiving statins, antihypertensive, and antiplatelet medications differ between genders [6,7].

In Spain, a recent observational, prospective study reported that women with T2DM have threefold higher odds of CV death than men [8]. Additionally, previously published cross-sectional and population-based studies indicated a poorest control of CVRF in primary and secondary prevention among Spanish women [9–12]. In all of these studies, the proportion of women was substantially lower than men, and, most importantly, the baseline characteristics differed significantly between cohorts. For instance, women were on average 2.5–4 years older than men, the duration of T2DM was nearly 1 year longer, they were less likely to smoke, and the prevalence of diabetes-related micro- and macrovascular complications was different between genders. Although these and other differences largely exist in real-life clinical practice, they may limit the interpretation of research findings when traditional cohort matching strategies, stratified analyses, or regression covariate adjustments are used to consider heterogeneity [13]. In contrast, when patients are matched with propensity modeling technologies, the cohorts have a balanced distribution of covariates, thus allowing for equivalent comparisons between groups that can provide inferences about causal effects in observational studies [13].

In Catalonia (Spain), the healthcare system is public and universal. The primary care centers provide first contact and continuing care for persons with any health concerns, and they are usually the principal place where T2DM is diagnosed and managed. The antidiabetic treatment is free of charge for those retired and severely ill people, while active subjects pay just a small part of the cost of the drugs [14]. Briefly, the primary care physicians are responsible for prescribing medications through an electronic prescription that the patient can pick up at the pharmacy. To assess prescribing practices concerning the appropriate use of drugs, the Health Institute of Catalonia uses a quality indicator system created in 2003, the Pharmaceutical Prescription Quality Standard (EQPF) [15]. This study aimed to evaluate whether the pharmacological management of T2DM and the degree of CVRF control in primary care differ between sexes in primary and secondary prevention using a propensity score matching method to balance the inequality of confounding covariates.

### **2. Materials and Methods**

### *2.1. Study Design*

This was a matched, cross-sectional study including data from patients with T2DM available from the SIDIAP population-based database. This database contains anonymized patient information from the computerized medical records stored in the Electronic Clinical station in Primary Care (eCAP). SIDIAP includes data from about 80% of the Catalonia population (5.835 million subjects) distributed within the 279 primary care centers belonging to the Catalan Health Institute (ICS) [16]. The overall T2DM population has been previously described [17], and this dataset was further used to apply the propensity score method.

The investigation conformed with the principles outlined in the Declaration of Helsinki. The study was approved by the Ethics Committee of the Primary Healthcare University Research Institute (IDIAP) Jordi Gol (P14/018) and registered at ClinicalTrials.gov (NCT04653805).

### *2.2. Study Variables*

We used data extracted data from patients aged 31 to 90 years with a diagnosis of T2DM (International Classification of Disease 10 [ICD-10] codes E11 and E14) as at 30 June 2013 who had at least one visit registered with the primary care team in the previous 12 months. For this study, the following variables were used: age, gender, time since diagnosis (years), smoking habit, number of visits with the primary care team in the previous 12 months, estimated glomerular filtration rate (eGFR) with the MDRD (modification of diet in renal disease) formula, presence of diabetic retinopathy (ICD-10 codes E11.3 and H36.0), albumin/creatinine ratio, BMI, glycated hemoglobin (HbA1c), lipid profile (i.e., total cholesterol levels, high-density lipoprotein cholesterol (HDL-c), lowdensity lipoprotein cholesterol (LDL-c), and triglycerides (TGs)), presence of dyslipidemia (defined as receiving medication for this condition), prescription of glucose-lowering drugs, lipid-lowering drugs (statins or other), blood pressure (BP) (diastolic (dBP) and systolic (sBP)), hypertension (defined as receiving medication for this condition), prescription of hypertension-lowering drugs, and antiplatelet and anticoagulant therapy. Chronic kidney disease was assumed in patients with eGFR < 60 mL/min and/or albumin/creatinine ratio > 300 mg/g. The most recent value registered was used in all cases. For those with a previous CVD, diagnostic codes for macrovascular diseases were collected, including coronary artery disease (CAD; ICD-10 codes I20-I24), cerebrovascular disease (ICD-10 codes I63, I64, G45 or G46), and peripheral artery disease (PAD; ICD-10 code 173.9).

Variables to assess the degree of CVRF control and treatment goals achievement were based on local guidelines [18], i.e., HbA1c ≤ 7% (53 mmol/mol), BP ≤ 140/90 mmHg, and LDL-c < 130 mg/dL (3.37 mmol/L) for primary prevention and <100 mg/dL (2.59 mmol/L) for secondary prevention. Additionally, the same variables were assessed according to the threshold stated by our institution (ICS): HbA1c ≤ 8% (64 mmol/mol), BP ≤ 130/80 mmHg, and LDL-c < 100 mg/dL for primary prevention and LDL-c < 70 mg/dL (1.81 mmol/L) for secondary prevention.

### *2.3. Propensity Score Matching Method*

Propensity score matching (PSM) was used to create subpopulations of women and men with T2DM that were balanced in terms of baseline conditions, namely, age, duration of T2DM, number of visits to the primary care team, presence of comorbidities (i.e., hypertension, dyslipidemia, and diabetic retinopathy), eGFR value, albumin/creatinine ratio, and smoking in primary prevention. For the analyses of those in secondary prevention, subjects were also matched for previous macrovascular diseases. Matched groups (male versus female group) were performed (1:1) using the one-to-one nearest neighbor algorithm (with a caliper of 0.1 of the SD of the propensity score on the logit scale) and no replacement. To evaluate PSM quality, we assessed the balance in covariates comparing the absolute difference before and after the matching procedure.

### *2.4. Statistical Analysis*

We summarized data as the mean (standard deviation) for continuous variables and number (percentage) for categorical variables by groups. To assess the association between clinical variables and gender, we computed the absolute difference in the means or proportions (Dif) between groups, and we estimated their 95% confidence interval (95% CI). To assess the magnitude of the gender differences, we calculated the relative percentage difference (rDif) between groups. Dif was calculated by subtracting the mean or proportion for women from the mean or proportion for men, and rDif was calculated as the absolute difference divided by the reference value (mean or proportion value of men) multiplied by 100. We performed graphical analyses with smoothing line plots to evaluate whether the potential differences remained over all age ranges. We performed a complete-case analysis excluding missing information for each quantitative variable. All analyses were performed using the R free software environment for statistical computing (v3.5.1) and the "MatchIt" library for the PSM [19].

### **3. Results**

A total of 343,969 patients with T2DM were identified in the database. After the matching procedure, there were 70,453 subjects in each primary prevention group and 19,593 in each secondary prevention group (Figure 1). Baseline characteristics in these populations were well balanced (Figures S1 and S2).

**Figure 1.** Flow chart of the propensity score matching procedure.

### *3.1. Primary Prevention*

The baseline characteristics of the matched women and men in primary prevention are shown in Table 1. The mean age of the overall population was 66.2 years (SD = 12.2), and the mean duration of diabetes 7.1 years (SD = 5.4) years. Dyslipidemia was present in 52.3% of the patients, hypertension was present in 66.7% of the patients, diabetic retinopathy was present in 6.3% of the patients, and renal impairment was present in 15.9% of the patients.

**Table 1.** Baseline characteristics of matched women and men with T2DM in primary prevention by gender.

95% CI, 95% confidence interval; BMI, body mass index; dBP, diastolic blood pressure; HbA1c, glycated hemoglobin; HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; Dif, difference between groups; sBP, systolic blood pressure; SD, standard deviation; TGs, triglycerides. \* Variables matched between study groups. † Number of visits with the primary care team in the previous 12 months. \$ Renal disease, including eGFR < 60 mL/min and/or albumin/creatinine ratio > 300 mg/g.

In this primary prevention population, women had higher BMI than men (Dif = 1.75 kg/m2; 95% CI = 1.7 to 1.8) but similar values of HbA1c (Dif = 0.02%; 95% CI = 0.01 to 0.03) and BP (dBP Dif = −0.49 mmHg; 95% CI= −0.5 to −0.4 and sBP Dif = −1.03 mmHg; 95% CI = −1.1 to 0.9). Although the plasmatic TG concentration was comparable between genders, total cholesterol, HDL-c, and LDL-c were higher in women than men (Dif = 12.13 mg/dL, 95% CI = 11.9 to 12.3; Dif = 6.23, 95% CI = 6.1–6.3; Dif = 5.50 mg/dL, 95% CI = 5.3 to 5.7, respectively). Moreover, this sex-difference in total cholesterol and LDL-c was observed across all age ranges (Figure 2A).

**Figure 2.** Smoothing line charts with changes in LDL-c, total cholesterol, and statin treatment across age in subjects on primary prevention (**A**) and secondary prevention (**B**) by gender (LDL-c, low-density lipoprotein cholesterol).

Differences by gender in the pharmacological management of T2DM and degree of CVRF control are shown in Table 2 and Figure 3. As for lipid control, statins were more frequently prescribed to women (rDif = 4.7%; Figure 3A). Regarding BP treatment, the prescription of diuretics, beta-blockers, and two antihypertensive drugs was substantially higher in women relative to men (rDif = 16.5%, 10.3%, and 8.1%, respectively). Lastly, women received antiplatelet therapy less often than men (rDif = −15.0%).

**Table 2.** Pharmacological treatment and cardiovascular risk factor control in matched women and men with T2DM in primary prevention by gender.

**Table 2.** *Cont.*

95% CI, 95% confidence interval; ACEI/ARBII, angiotensin-converting enzyme inhibitors/angiotensin II receptor blockers; BP, blood pressure; CCB, calcium channel blockers; CVRF, cardiovascular risk factor; HbA1c, glycated hemoglobin; LDL-c, low-density lipoprotein cholesterol; Dif, difference between groups. \* Lipid-lowering treatment, proportion data calculated on the basis of those with dyslipidemia. † Antihypertensive treatment, proportion data calculated on the basis of those with hypertension.

The proportion of women who achieved BP target levels was greater in women for both the ≤130/80 mmHg and the ≤140/90 mmHg goals (rDif = 8.8% and 2.5%, respectively). Despite women being more frequently treated with statins than men, fewer women attained the LDL-c ≤ 130 and ≤100 mg/dL thresholds relative to men (rDif = −7.5% and rDif = −14.8%, respectively) (Figure 3B). Regarding glycemic control, the gender differences in the proportion of subjects below the HbA1c ≤ 7 and 8% target was negligible (rDif = −0.7% and 0.1%, respectively). Lastly, the combined achievement of HbA1c, BP, and LDL-c goals was poorest in women relative to men (rDif = −7.4% for LDL-c target < 130 mg/dL and rDif = −14.9% for target ≤ 100 mg/dL).

### *3.2. Secondary Prevention*

Baseline characteristics of the matched women and men with T2DM in secondary prevention are shown in Table 3. Overall, subjects were 74.9 years old (SD = 9.9) with a mean diabetes duration of 9.3 years (SD = 6.4). A significant proportion of patients had dyslipidemia (78.8%), and almost all had hypertension (92.6%). Moreover, 11.7% and 35.6% of subjects presented diabetic retinopathy and renal impairment, respectively. Regarding macrovascular diseases, CAD was the most common prior complication (59.9%), followed by cerebrovascular disease (37.6%) and PAD (13.9%).

**Figure 3.** Plot of the relative percent difference between genders for treatments prescribed (**A**) and target achievement (**B**) in the population in primary prevention (ACEI/ARBII, angiotensinconverting enzyme inhibitors/angiotensin II receptor blockers; BP, blood pressure; CCB, calcium channel blockers; HbA1c, glycated hemoglobin; LDL-c, low-density lipoprotein cholesterol; OAD, oral antidiabetic drug).

**Table 3.** Baseline characteristics of matched women and men with T2DM in secondary prevention by gender.

95% CI, 95% confidence interval; BMI, body mass index; CAD, coronary artery disease; dBP, diastolic blood pressure; Dif, difference of means between groups; HbA1c, glycated hemoglobin; HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; PAD, peripheral artery disease; rDif, relative percentage difference between sexes; sBP, systolic blood pressure; SD, standard deviation; TGs, triglycerides. \* Variables matched between study groups. † Number of visits with the primary care team in the previous 12 months. \$ Renal disease, including eGFR < 60 mL/min and/or albumin/creatinine ratio > 300 mg/g.

Similar to what was observed in primary prevention patients, women had higher BMI than men (Dif = 1.69 kg/m2, 95% CI = 1.6 to 1.8) but there were no clinically significant differences in HbA1c levels (Dif = 0.11%, 95% CI = 0.09 to 0.1) and BP values (dBP Dif = 0.53 mmHg, 95% CI = 0.4 to 0.6; sBP Dif = 1.00 mmHg, 95% CI = 0.9 to 1.1). Regarding the lipid profile, TG levels in women were comparable to those observed in primary prevention while they were considerably lower in men, which widened the difference between genders (Dif = 11.34 mg/dL; 95% CI = 10.3 to 12.4). All other parameters, such as total cholesterol, HDL-c, and LDL-c were lower than those observed in primary prevention subjects, particularly in men, and all substantially higher among women (Dif = 16.89 mg/dL; 95% CI = 16.5 to 17.3; Dif = 5.77, 95% CI = 5.6 to 5.9; Dif = 8.42 mg/dL; 95% CI = 8.1 to 8.8, respectively). As shown in Figure 2B, these higher total cholesterol and LDL-c levels in women were observed from 40 years onward and persisted in all age groups. In comparison, values in men progressively decreased until around 80 years of age.

Differences by gender in the pharmacological management of T2DM and degree of CVRF control are shown in Table 4 and Figure 4. The proportion of patients prescribed statins was similar between genders (rDif = −0.5%), but treatment with diuretics and three antihypertensive drugs was more frequent in women relative to men (rDif = 18.5% and rDif = 5.3%, respectively) (Figure 4A). Moreover, women received less often antiplatelet and anticoagulant therapy (rDif = −5.7% and −4.0%, respectively). Although the proportion of patients treated with glucose-lowering drugs was similar between groups (rDif = −1%), women were less often prescribed one or more oral antidiabetic drugs (OAD) than men (rDif = −3.8% for one and −18.6% for more than one OAD). Moreover, women were more frequently treated with either insulin alone (rDif = 19.6%) or combined with one or more OAD (rDif =32.2% with one OAD and 8.2% with more than one OAD).

**Table 4.** Pharmacological treatment and cardiovascular risk factor control of matched women and men with T2DM in secondary prevention by gender.

95% CI, 95% confidence interval; ACEI/ARBII, angiotensin-converting enzyme inhibitors/angiotensin II receptor blockers; BP, blood pressure; CCB, calcium channel blockers; CVRF, cardiovascular risk factor; Dif, difference between groups; HbA1c, glycated hemoglobin; LDL-c, low-density lipoprotein cholesterol; OAD, oral antidiabetic drug; rDif, relative percentage difference between sexes. \* Lipid-lowering treatment, proportion data calculated on the basis of those with dyslipidemia. † Antihypertensive treatment, proportion data calculated on the basis of those with hypertension.

**Figure 4.** Plot of the relative percentage difference between genders for treatments prescribed (**A**) and target achievement (**B**) in the population in secondary prevention (ACEI/ARBII, angiotensinconverting enzyme inhibitors/angiotensin II receptor blockers; BP, blood pressure; CCB, calcium channel blockers; HbA1c, glycated hemoglobin; LDL-c, low-density lipoprotein cholesterol; OAD, oral antidiabetic drug).

Despite women receiving more intensive antihypertensive treatment, they achieved BP control less frequently than men, either at the ≤130/80 mmHg or at the ≤140/90 mmHg goal (rDif = −6.9% and rDif = −4%, respectively) (Figure 4B). Although the proportion of patients prescribed lipid-lowering treatments was similar between sexes, the targets LDL < 100 mg/dL and <70 mg/dL were less often reached among women (rDif = −16.8% and rDif = −27.7%). Regarding glycemic goals, women showed slightly worse control relative to men (rDif = −5.0% for HbA1c ≤ 7% and −3.5% for HbA1c ≤ 8%). In accordance, the combined target goals of glycemia (HbA1c ≤ 7%), blood pressure (BP ≤ 140/90 mmHg), and LDL-c were less frequently achieved by women than men (rDif = −21.6% for LDL-c < 100 mg/dL and rDif = −29.9% for LDL-c < 70 mg/dL).

### **4. Discussion**

The results of this propensity score-matched analysis in patients with T2DM showed that both genders exhibited comparable BP and HbA1c levels, but women had higher BMI and a significantly poorer lipid profile than men. Moreover, there were sex disparities in treatment prescription. Women were more frequently above recommended treatment goals, particularly LDL-c, and the worst overall CVRF control was more pronounced in secondary prevention patients.

The disparities in baseline characteristics between groups were observed in both the primary and secondary prevention cohorts and agree with previous observational studies conducted in Spain and other international large cohort studies assessing sex differences in T2DM risk and management [2,9–12,20]. Indeed, it has been estimated that women have a BMI nearly 2 kg/m2 higher than men at T2DM diagnosis despite similar levels of HbA1c [21,22]. This discrepancy has mainly been attributed to the physiological fat distribution in women, which is characterized by more subcutaneous fat mass and less liver and visceral fat, in addition to greater glucose sensitivity compared with men [20]. Thus, women need to gain more weight and accumulate adiposity to establish a diagnosis of diabetes, which extends the prediabetes state with a result of impairment of CVRF [23].

In our study, women had a worse overall lipid profile relative to men, mainly from approximately 40 years onward. These results agree with recent studies reporting that women with T2DM, particularly after menopause, have higher total cholesterol, LDL-c, and HDL-c than men with T2DM [19,24]. This disparity would lead to a more atherogenic lipid and proinflammatory profile in women with T2DM, in turn linked with an increased cardiometabolic risk [25].

Most notably, our findings confirm inequalities between genders in the pharmacological treatment of T2DM and the ability to reach guideline-recommended targets [2]. The unfavorable lipid profile and difficulties in reaching LDL-c levels below treatment goals among T2DM women regardless of statin treatment are well documented [10,12,19,26]. Although the prescription of statins in our study was slightly more frequent in women in primary prevention and used at similar rates in both genders in secondary prevention, a considerably higher proportion of women were not able to reach the corresponding LDL-c targets relative to men in either condition. One explanation for this disparity could be that women are less likely to receive high-intensity statins than men [27,28]. Other factors may interfere, such as an inadequate adherence to statins (estimated to be 10% greater in women than in men [29]), worse tolerance to this drug class, and less likelihood than men to believe that statins are safe or effective [28].

Although there are divergences in the literature, most studies reported no differences between sexes regarding HbA1c control [2]. Our findings show that the degree of glycemic control was similar in both groups in primary prevention, but it was a little worse among women in secondary prevention. However, women were more likely to be prescribed insulin, alone or in combination. A large population-based study conducted in 415,294 Italian patients with T2DM reported that insulin was more frequently used in women than men when off the HbA1c target [26]. Moreover, that study found a wider use of diuretics in women than men and a slightly higher likelihood of reaching the BP

target < 130/80 mmHg [26]. We also found that women received more intense antihypertensive treatment, particularly diuretics, but they were more frequently on BP target only in the case of primary prevention. This agrees with a previous study conducted in our population [10], but contrasts two observational studies conducted in the Netherlands, where stratified analyses found no gender differences in the percentage of patients with or without CVD receiving antihypertensive medication and attaining BP control [20,30]. The discrepancy between studies may be more related to sociodemographic factors than sex-specific differences. For instance, one of the Dutch studies found that women with lower educational level had a higher likelihood of receiving antihypertensive medication when systolic BP > 140 mmHg and were at a higher CVD risk than men [20].

As a result of the suboptimal management of individual CVRF among women (particularly LDL-c levels in primary prevention, and LDL-c and BP levels in secondary prevention), the simultaneous attainment of glucose, lipid, and BP recommended goals was considerably less satisfactory among women even when more intensely treated than men. It has been reported that this gap in CV risk burden is due to the existence of additive factors beyond biological dissimilarities, such as lifestyle, cultural and/or socioeconomic factors, and physician biases [2]. For instance, physical activity levels are lower in women with T2DM than their male counterparts [31], and men were more successful in reducing and maintaining weight than women in most studies [32]. Furthermore, there is still a widespread belief among health professionals that CVD is more prevalent in men, leading to underestimation of the problem among women and, consequently, to undertreatment [23]. Moreover, the intensified multifactorial treatment approach, including nonpharmacological (lifestyle recommendations and close monitoring of laboratory and clinical parameters) and pharmacological treatment, have demonstrated a remarkable benefit for reducing the risk of major cardiovascular events (MACEs) and mortality in high-risk diabetic kidney disease [33]

The main strength of this study is the use of real-world data from a large dataset of primary healthcare services in Catalonia that includes urban and rural areas. Moreover, we used a propensity score matching method to homogenize the sample with a satisfactory reduction in absolute differences of potential confounding variables between genders after the matching procedure. Some studies examined the performance of several methods using PSM for the estimation of different measures of association, showing that the PSM approach estimates with less bias than other regression techniques [34–36]. However, the findings of this study must be seen in light of some limitations. Firstly, the cross-sectional design did not allow establishing a causal relationship between the variables. Secondly, we had no data on variables known to contribute to the observed sex dimorphism in diabetes risk and outcome, such as psychosocial risk factors (e.g., socioeconomic status, social support, or educational level) or health behavior (e.g., diet, physical activity, alcohol consumption). Thirdly, we had no data on the doses of the prescribed drugs and whether there were any contraindications (allergies, comorbidities, etc.) that could partially explain gender differences in the disease management. Moreover, we could not assess adherence to the prescribed medications, which may have partly contributed to the observed disparity in CVRF control between sexes. Fourthly, it is not known which comes first, the specific laboratory result (total cholesterol, LDL-c, HDL-c, TGs, and HbA1c) or the particular drug prescription. However, this bias would be present in both groups. Moreover, we did not use the CV risk classification from the 2019 ESC/EASD Guidelines (i.e., moderate, high, or very high CV risk) as the data used predated this recommendation, and the applicable stratification at that time was the requirement of primary vs. secondary prevention. A large population-based study conducted on 373,185 type 2 diabetic subjects in Catalonia reported that at least 50% of them were at very high risk of CV events according to ESC/EASD 2019 classification, and approximately 26% presented with previous CVD [37]. This figure is similar to the proportion of subjects with prior CVD that we included in the secondary prevention group in our study (21.8%). However, categorizing and treating patients according to their CV risk as per the new recommendations will probably provide a more comprehensive and tailored T2DM management than if we only consider the

primary/secondary approach [38]. Lastly, we cannot discard that the physician's sex might have somehow influenced the patient's assessment and care.

### **5. Conclusions**

It is essential that, in the process of care, healthcare professionals, from nurses to physicians and researchers, know and consider that CVD is not only a male issue. Inequalities in the management and control of CVRF in women with T2DM may contribute to an increased risk of CVD compared with men. While more research is needed to elucidate the causes of these inequalities, there is a need to implement gender-sensitive strategies to minimize the existing treatment gap. These should include more stringent follow-up implementing an intensified multifactorial treatment approach to achieve optimal risk factor management and educational programs for healthcare professionals and patients to give visibility and cope with gender disparities.

**Supplementary Materials:** The following supporting information can be downloaded at https:// www.mdpi.com/article/10.3390/jcm11082196/s1: Figure S1. Absolute differences between patients with T2DM according gender in primary prevention (DaysDM, days since type 2 diabetes mellitus diagnosis; Smoke: n/d, smoking status not known; ExSmoke, former smoker; HTA, hypertension; DSL, dyslipidemia; Nvis\_m, number of visits done in primary healthcare last year; FG\_cat62, glomerular filtration rate < 15 mL/min; FG\_cat63, glomerular filtration rate 15–30 mL/min; FG\_cat64, glomerular filtration rate 31–44 mL/min; FG\_cat65, glomerular filtration rate 45–59 mL/min; FG\_cat69, glomerular filtration rate > 60 mL/min; QAC\_cat2, urinary albumin creatinine ratio < 30 mg/g; QAC\_cat3, urinary albumin creatinine ratio 30–300 mg/g; QAC\_cat4, urinary albumin creatinine ratio > 300 mg/g; RD, diabetic retinopathy); Figure S2. Absolute differences between patients with T2DM according gender in secondary prevention (DaysDM, days since type 2 diabetes mellitus diagnosis; Smoke: n/d, smoking status not known; ExSmoke, former smoker; HTA, hypertension; DSL, dyslipidemia; Nvis\_m, number of visits done in primary healthcare last year; FG\_cat62, glomerular filtration rate < 15 mL/min; FG\_cat63, glomerular filtration rate 15–30 mL/min; FG\_cat64, glomerular filtration rate 31–44 mL/min; FG\_cat65, glomerular filtration rate 45–59 mL/min; FG\_cat69, glomerular filtration rate > 60 mL/min; QAC\_cat2, urinary albumin creatinine ratio < 30 mg/g; QAC\_cat3, urinary albumin creatinine ratio 30–300 mg/g; QAC\_cat4, urinary albumin creatinine ratio > 300 mg/g; RD, diabetic retinopathy; artper, peripheral artery disease; ci, coronary disease; avc, cerebrovascular disease; ic, heart failure).

**Author Contributions:** Conceptualization, D.M. and J.F.-N.; methodology, D.M., J.F.-N., J.R. and A.R.-M.; data curation, J.R. and A.R.-M.; investigation, D.M., J.F.-N., J.R. and A.R.-M.; formal analysis, J.R.; writing—original draft preparation, A.R.-M.; writing—review and editing, D.M., J.F.-N., J.R., M.G. and A.R.-M.; validation, D.M., J.F.-N., J.R., M.G. and A.R.-M.; visualization, D.M., J.F.-N., J.R., M.G. and A.R.-M.; supervision, D.M. and J.F.-N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The investigation conformed with the principles outlined in the Declaration of Helsinki. The study was approved by the Ethics Committee of the Primary Healthcare University Research Institute (IDIAP) Jordi Gol (P14/018) and registered in the ClinicalTrials.gov (NCT04653805).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available from the corresponding author upon reasonable request.

**Acknowledgments:** The authors acknowledge Helena Kruyer for language editing and proofreading and the Territorial Management of Central Catalonia of Institut Català de la Salut (ICS) for the grant for research training and doctoral completion in primary healthcare.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

## *Article* **Development of a Complex Intervention for Effective Management of Type 2 Diabetes in a Developing Country**

**Tigestu Alemu Desse 1,\*, Kevin Mc Namara 2,3, Helen Yifter 4 and Elizabeth Manias 1**

**Abstract:** There has been little focus on designing tailored diabetes management strategies in developing countries. The aim of this study is to develop a theory-driven, tailored and context-specific complex intervention for the effective management of type 2 diabetes at a tertiary care setting of a developing country. We conducted interviews and focus groups with patients, health professionals, and policymakers and undertook thematic analysis to identify gaps in diabetes management. The results of our previously completed systematic review informed data collection. We used the United Kingdom Medical Research Council framework to guide the development of the intervention. Results comprised 48 interviews, two focus groups with 11 participants and three co-design panels with 24 participants. We identified a lack of structured type 2 diabetes education, counselling, and collaborative care of type 2 diabetes. Through triangulation of the evidence obtained from data collection, we developed an intervention called VICKY (patient-centred collaborative care and structured diabetes education and counselling) for effective management of type 2 diabetes. VICKY comprised five components: (1) patient-centred collaborative care; (2) referral system for patients across transitions of care between different health professionals of the diabetes care team; (3) tools for the provision of collaborative care and documentation of care; (4) diabetes education and counselling by trained diabetes educators; and (5) contextualised diabetes education curriculum, educational materials, and documentation tools for diabetes education and counselling. Implementation of the intervention may help to promote evidence-based, patient-centred, and contextualised diabetes care for improved patient outcomes in a developing country.

**Keywords:** type 2 diabetes; complex intervention; behaviour change intervention; co-design; continuity of care; developing country; Ethiopia; patient participation; patient transfer

### **1. Introduction**

Type 2 diabetes is a global public health problem and an economic burden to nations, particularly developing countries [1]. It contributes to cardiovascular complications, such as ischemic heart disease, heart failure, and renal disorders [2–4].

Ineffective management of type 2 diabetes has been associated with poor clinical outcomes, which include disease progression, and increased health services utilisation, such as repeated hospitalisations and high all-cause mortality [5–7]. In Sub-Saharan Africa (SSA) [8–10] including Ethiopia [11–13], there exists a high rate of diabetes-related morbidity and mortality, high cost of diabetes care, and poor quality of life for patients with type 2 diabetes. Excessive levels of diabetes-related problems and high cost of type 2 diabetes care in SSA are attributed to widespread lack of treatment success, stemming from inadequate organisational involvement

**Citation:** Desse, T.A.; Namara, K.M.; Yifter, H.; Manias, E. Development of a Complex Intervention for Effective Management of Type 2 Diabetes in a Developing Country. *J. Clin. Med.* **2022**, *11*, 1149. https://doi.org/ 10.3390/jcm11051149

Academic Editor: Fernando Gómez-Peralta

Received: 24 January 2022 Accepted: 18 February 2022 Published: 22 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and delivery of care [8,9,14]. Multiple contributing factors exist in the region, such as a lack of contextually tailored diabetes management approaches, inadequate diabetes training of health professionals, and low levels of collaborative care and effective shared treatment plans developed by patients and health professionals [10,14–16]. In Ethiopia, similar factors contributing to ineffective management of type 2 diabetes exist, including inadequate collaborative care among pharmacists, physicians, and nurses; lack of structured diabetes education; and high levels of medication therapy problems and diabetes complications [13,17–23].

Patient-centred collaborative care and the use of culturally tailored interventions, including behavioural interventions, can improve diabetes care in low-income countries [14,24,25]. Evidence indicates that SSA nations require evidence-based type 2 diabetes management strategies tailored to the context and aimed at reducing diabetes-related morbidity and mortality and high healthcare costs [10,16,26]. However, there has been little focus on designing contextually tailored type 2 diabetes management strategies in this region. While evidence suggests that structured diabetes education and counselling and collaborative care by pharmacists, physicians, nurses, and other health professionals can improve health outcomes and cost of type 2 diabetes treatment [27–30], implementation needs for such elements of care are not readily understood for type 2 diabetes in Ethiopia [21,22,31,32]. Furthermore, studies examining type 2 diabetes in Ethiopia are mainly observational and focused on the rate of glycemic control, magnitude of diabetesrelated complications, quality of care, and mortality. Moreover, there has been no focus on designing appropriately tailored interventions to improve diabetes care [12,13,18,33–38]. To the authors' knowledge, there has been no pragmatic study undertaken to explore the dynamics of current management for type 2 diabetes at a micro- or meso-level or devise much-needed diabetes management strategies tailored to SSA [10,14,16,39,40].

The aim of this study was to develop a theory-driven, tailored, and context-specific complex intervention for the effective management of type 2 diabetes at a tertiary care setting of a developing country.

### **2. Materials and Methods**