*Article* **Proangiogenic Effect of 2A-Peptide Based Multicistronic Recombinant Constructs Encoding VEGF and FGF2 Growth Factors**

**Dilara Z. Gatina 1, Ekaterina E. Garanina 1, Margarita N. Zhuravleva 1, Gulnaz E. Synbulatova 1, Adelya F. Mullakhmetova 1, Valeriya V. Solovyeva 1, Andrey P. Kiyasov 1, Catrin S. Rutland 2, Albert A. Rizvanov 1,\* and Ilnur I. Salafutdinov 1,\***

**Citation:** Gatina, D.Z.; Garanina, E.E.; Zhuravleva, M.N.; Synbulatova, G.E.; Mullakhmetova, A.F.; Solovyeva, V.V.; Kiyasov, A.P.; Rutland, C.S.; Rizvanov, A.A.; Salafutdinov, I.I. Proangiogenic Effect of 2A-Peptide Based Multicistronic Recombinant Constructs Encoding VEGF and FGF2 Growth Factors. *Int. J. Mol. Sci.* **2021**, *22*, 5922. https:// doi.org/10.3390/ijms22115922

Academic Editors: Paul Quax and Elisabeth Deindl

Received: 29 April 2021 Accepted: 27 May 2021 Published: 31 May 2021

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**Copyright:** © 2021 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/).

**Abstract:** Coronary artery disease remains one of the primary healthcare problems due to the high cost of treatment, increased number of patients, poor clinical outcomes, and lack of effective therapy. Though pharmacological and surgical treatments positively affect symptoms and arrest the disease progression, they generally exhibit a limited effect on the disease outcome. The development of alternative therapeutic approaches towards ischemic disease treatment, especially of decompensated forms, is therefore relevant. Therapeutic angiogenesis, stimulated by various cytokines, chemokines, and growth factors, provides the possibility of restoring functional blood flow in ischemic tissues, thereby ensuring the regeneration of the damaged area. In the current study, based on the clinically approved plasmid vector pVax1, multigenic constructs were developed encoding vascular endothelial growth factor (VEGF), fibroblast growth factors (FGF2), and the DsRed fluorescent protein, integrated via picornaviruses' furin-2A peptide sequences. In vitro experiments demonstrated that genetically modified cells with engineered plasmid constructs expressed the target proteins. Overexpression of VEGF and FGF2 resulted in increased levels of the recombinant proteins. Concomitantly, these did not lead to a significant shift in the general secretory profile of modified HEK293T cells. Simultaneously, the secretome of genetically modified cells showed significant stimulating effects on the formation of capillary-like structures by HUVEC (endothelial cells) in vitro. Our results revealed that when the multicistronic multigene vectors encoding 2A peptide sequences are created, transient transgene co-expression is ensured. The results obtained indicated the mutual synergistic effects of the growth factors VEGF and FGF2 on the proliferation of endothelial cells in vitro. Thus, recombinant multicistronic multigenic constructs might serve as a promising approach for establishing safe and effective systems to treat ischemic diseases.

**Keywords:** angiogenesis; gene expression; non-viral vectors; 2A-peptides; growth factors; tube formation; VEGF; FGF2; cytokines

### **1. Introduction**

Currently, ischemic diseases remain one of the leading causes of death in the world's developed countries [1]. This disease group is characterized by a lack of oxygen and nutrient supply due to impaired micro- and macro-blood supply to a tissue, organ, or limb [2]. Simultaneously, the lack of an adequate blood supply stimulates the activation of angiogenesis processes due to the release of proangiogenic factors [3]. In this regard, a strategy of supportive angiogenic therapy was proposed, which underlies the introduction of exogenous growth factors that promote vasculogenesis and blood circulation restoration [4].

Angiogenesis depends on complex interactions including spatial-temporal interaction of cells and various proangiogenic factors, particularly vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF2). As the most critical angiogenic factor, vascular endothelial growth factor (VEGF) remains one of the promising candidates for proangiogenic therapy [5–7]. The results of numerous in vitro and in vivo experiments have demonstrated that VEGF promotes de novo new blood vessel formation, improves blood flow, and supports myogenesis [8,9]. In addition, binding to the VEGFR1 and VEGFR2 receptors increases vascular permeability and induces proliferation and migration of endothelial cells [10]. It is known that VEGF contributes to the survivability of endothelial cells preventing apoptosis via the PI3K/AKT-signaling pathway and induces expression of the antiapoptotic proteins A1 and Bcl-2 [11–13]. The crucial role of VEGF in angiogenic processes has been confirmed in numerous investigations. The knockout of even one allele of the VEGF gene led to embryonic lethality and disorders in the cardiovascular system [14]. VEGF-A inhibitors are widely used in the therapy of solid tumors, thus indicating the significant role of VEGF in both normal and pathological angiogenesis [15]. Over the last decade, several randomized controlled trials have also utilized plasmid and viral vectors with the VEGF gene to treat severe coronary heart disease (Euroinject One, KAT, REVASC, NOTHERN, NOVA, VEGF-A Neupogen, and GENASIS) [6,16]. In 2011, the drug Neovasculgen was approved in Russia (ClinicalTrials.gov identifier: NCT03068585) to treat lower limb ischemia of atherosclerotic genesis, including chronic critical lower limb ischemia. This drug represents a highly purified, supercoiled form of the plasmid pCMV-VEGF165-encoding isoform 165 (a) of vascular endothelial growth factor [17,18].

The basic fibroblast growth factor (FGF2) is the second of the most characterized mitogens utilized in gene therapy protocols to induce therapeutic angiogenesis [19]. FGF2 interacts with the FGFR1 and FGFR2 receptors and with heparan sulfate proteoglycans, which results in proliferation and migration of endothelial cells, protease production, and angiogenesis. Proangiogenic effects of FGF2 have been confirmed in numerous experimental in vivo models of angiogenesis, including the chick embryo chorioallantoic membrane model, the cornea of the eye, and matrigel implants [20]. Moreover, positive effects of FGF2 have been reported in cases of pressure sores, diabetic foot ulcers, and burns. In 2001, Trafermin, a medicine based on human FGF2 for treating pressure ulcers and skin ulcers, was approved in Japan [21]. Likewise, the application of FGF2 has been successful in the treatment of second-degree burns. In the randomized study, patients with deep burns received local injections of either a placebo or bovine FGF2. All patients treated with FGF2 demonstrated faster granulation tissue formation and epidermal regeneration than patients in the placebo group [22]. Similar to VEGF, FGF2 has an affinity for heparin, increases neuronal survival, and reduces apoptotic cell death [23,24]. Unlike VEGF, FGF2 is capable of stimulating the proliferation of not only endothelial cells but also smooth muscle cells and fibroblasts. FGF2 also regulates the recruitment of immune cells such as monocytes, macrophages, and neutrophils, affecting the production of various chemokines [25,26].

The synergetic effects of VEGF and FGF2 were demonstrated in a broad range of studies dedicated to induction of angiogenesis and blood flow restoration [27,28]. For example, it has been described that FGF2 can induce endogenic VEGF expression and its receptors in endothelial cells where VEGFR2 inhibitors had arrested FGF2-induced angiogenesis [29]. Numerous studies have also shown that coordinated activity of VEGF and FGF2 is crucial in all stages of angiogenesis, especially during early embryogenesis, to increase vascular permeability and recruitment of endothelial cells. Stimulation of endothelial cells initiates protease production and plasminogen secretion, destroying basement membrane and activating the invasion of cells in the surrounding matrix [30,31]. Though the exact synergetic mechanism remains unclear, there are several potential signaling pathways involved in both VEGF and FGF2 interactions and interactions with their receptors. Interaction between VEGF–VEGFR is known to activate the Ras-MEK-MAPK and AKT, P38, and PKC

signaling pathways, which in turn modulate FGF2 expression and proangiogenic cellular responses [32]. The interaction between FGF2–FGFR also activates Ras-MEK-MAPK signaling pathways and induces VEGF expression [33]. SRC cascade, AKT/P13K, and PKC pathways are known to participate in FGF2-dependent angiogenesis [34]. Thus, interaction between VEGF and FGF2 activate numerous signaling cascades and ultimately stimulate the angiogenic processes in endothelial cells.

Augmented concentrations of proangiogenic factors are known to cause aberrant vessel formation and hemangiomas as well [35]. These results were obtained when viral constructions were used providing longitudinal transgene expression [36]. It is common knowledge that viral vectors represent an efficient vehicle for target gen delivery due to their natural infection capacity of various cell types [37,38]. However, uncontrolled gene expression, immune responses to the constituent components of viral particles, and some viral genomes' relatively small packaging capacities restrict the feasibility of constructs based on recombinant viruses [39,40]. Compared to viruses, the application of non-viral gene therapy approaches using plasmid vectors do not provoke systemic reactions in the organism, and plasmids could be easily manufactured in preparative amounts [41]. Additionally, the development of new delivery systems has made it possible to achieve high efficiency of transfection both in vitro and in vivo. Continuous improvement of nonviral gene therapy techniques therefore contributes to their widespread use in clinical practice [9].

Currently, several strategies have been proposed to ensure simultaneous gene expression [42,43]: independent internal promoters [44], internal ribosomal entry sites (IRES) [45], mRNA splicing [46], bi-directional promoters, and 2A-peptides [47,48]. However, utilizing several promoters might significantly decrease transcriptional activity and reduce the vector's packaging capacity. In turn, IRESs provide effective co-expression of a few genes, but their large molecular weight (up to 1 kb) presents a limiting factor [49]. Moreover, the level of transgene expression depends on the location and spatial organization of the transgene. Downstream genes are transcribed less effectively in multicistronic constructions than cistrons located upstream of IRES [50–54].

Picormoviral 2A-sequences represent short peptides, composed of 14-21 amino acid residues, and contain a functional consensus motif Asp-Val/Ile-Glu-Asn-X-Pro-Gly -Pro [55]. This motif interrupts translation, "skipping" the formation of the last glycine-proline bond and releasing an upstream polypeptide that is attached to the C-terminal sequence of 2A. When translating the next polypeptide, proline is used as the first amino acid [56,57]. After 2A-mediated cleavage, the newly synthesized proteins contain a small N-terminal proline and a C-terminal residue of the 2A peptide; however, the proteins usually remain functionally active [57]. Currently, four types of 2A-peptide sequences are utilized in biomedical applications: foot-and-mouth disease virus FMDV 2A (F2A); equine rhinitis virus A (ERAV) 2A (E2A); porcine tospovirus 1 2A (P2A); and asigna virus 2A (T2A) [51]. Mostly, 2A-peptide dedicated studies focus on the efficiency of cleavage of various proteins in multicistronic vectors. For example, it has been demonstrated that T2A usually has the most efficient cleavage compared to other types of 2A-peptides. In contrast, other results have also revealed higher efficiencies of P2A-mediated cleavage in comparison to T2A [51,58]. It is quite important to consider post-translational conformation of target proteins to achieve the most effective transgene expression. For example, the secretion of TGF-β was arrested due to the coding sequence's upstream position to 2A peptide [59]. Incorporation of the furin cleavage site (Fu) upstream to the 2 sequence enables polyprotein convertase specifically to hydrolyze C-terminal peptide bonds of arginine and cleavage the proteins downstream of R-X-K/R-R motif. This sequence has been discovered in various human proteins, plays a crucial role in protein formation, and enables active secretion and membrane-associated proteins [60,61].

In the present study, using a clinically approved plasmid vector, we designed recombinant constructions containing picornavirus peptide sequences, codon-optimized sequences of VEGF and FGF2, and a reporter DsRed fluorescent protein under the control of a cytomegalovirus promoter. Expression of recombinant proteins, the secretome of modified cells, and the synergetic effects of the target proteins on the formation of capillary-like structures by HUVECs were investigated.

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

#### *2.1. Recombinant Constructs, Isolation, and Restriction Analysis of Plasmid DNA*

Based on a clinically approved plasmid vector pVax1 (Thermo Fisher Scientific) [62], multicistronic constructions encoding various gene combinations (pVax1-VEGF-FGF2- DsRed, pVax1-VEGF-DsRed, pVax1-FGF2-DsRed, pVax1-DsRed) were designed and developed (Figure 1). Nucleotide sequences of the VEGF (GeneBank AF486837.1) and FGF2 (GeneBank #DD406196.1) genes were obtained from the NCBI database and cloned under a single CMV promoter. The Fu-cleavage site (AGAAACAGAAGA) and p2A skipping motif (CCACGAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCT) were incorporated between the target genes [63]. To enhance gene expression and reduce restriction sites, an Optimum Gene™ (GenScript, Piscataway, NJ, USA) algorithm was applied. Synthesis of plasmid constructions de novo were carried out using GenScript (https://www.genscript.com/).

#### *2.2. Genetic Modification of the Cells with Recombinant Plasmids*

Human embryonic kidney cells HEK293T (ATCC CRL-11268) were cultivated on Dulbecco's modified media supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin/streptomycin, and 4 mM L-glutamine. A total of 2.5 × 105 cells were seeded per well in a 24-well plate in two ml medium. Transfections were carried once 70% confluence had been achieved using Turbofect transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA). Transfection efficiency was analyzed after 48 h using DsRed expression by the modified cells as an indicator using a fluorescent inverted micro-scope AxioObserver Z1 Carl Zeiss AG, Oberkochen, Germany).

#### *2.3. Quantitative Analysis of mRNA Expression*

Total RNA from genetically modified cells was isolated using TRIZOL (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer's instructions. Concentrations of isolated RNA were evaluated using a spectrophotometer NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized using a RevertAid kit (Thermo Fisher Scientific, Waltham, MA, USA), also in accordance with the recommended protocol. Expression of target genes was assessed by Real-time PCR and TaqMan technology on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Non-transfected 293 cells (NTC—non-transfected cells) served as negative controls for quantitative analysis of transgene expression. The relative amount of mRNA for the target genes was normalized to 18S rRNA and calculated using the 2−ΔΔCT. Nucleotide sequences of primers specific to codon-optimized sequences of target genes are presented in Table 1.

#### *2.4. Immunofluorescent Assays for Transfected HEK293T Cells*

Transfected HEK293T cells were primarily fixed with chilled methanol for 20 min at 20◦. Cell membranes were permeabilized by adding 0.1% Triton-100 (Helicon, Russia) and then incubated with primary antibodies at a dilution of 1:100, VEGF (Santa Cruze Biotechnology, CA, USA) and FGF2 (Santa Cruze Biotechnology, Santa Cruz, CA, USA), in Tris-buffer saline (TBS) for one hour. Following the primary antibody incubation period, the cells were washed with TBS and coated with the species-specific secondary. Nuclei were stained using a DAPI solution (4 ,6-diamidino-2-phenylindole;62248, Thermo Fisher Scientific, Waltham, MA, USA) and photomicrographs for analysis were captured on a fluorescent microscope AxioObserver Z1 (Carl Zeiss AG, Oberkochen, Germany).

**Table 1.** Nucleotide sequences of primers and probes used for qPCR.

#### *2.5. Enzyme-Linked Immunosorbent Assay (ELISA) of Target Genes*

The concentrations of VEGF and FGF2 in cell lysates and supernatants of transfected and native cells were evaluated using Human VEGF (DY293B) and Human FGF2 (DY233) kits (R&D Systems, DuoSet, Minneapolis, MN, USA) in accordance with the manufacturers' recommended protocol. The calibration curve was created based on seven different concentrations in the range of 31.1–2000 pg/mL. Optical densities were measured using BioRadxMark at OD 450. The resulting standard curve was utilized for quantitative analysis of the recombinant proteins.

#### *2.6. Multiplex Analysis*

Quantitative assessment of secreted soluble factors in conditioned media and cell lysates was measured using a Bio-Plex200 System. based on xMAP Luminex technology, with Bio-Plex Pro Human Cytokine 27-plex Assay (M500KCAF0Y) including the following analytes: IL-1, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, FGF2, Eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MP-1, MIP-1, MIP-1, PDGF-BB, RANTES, TNF-α, and VEGF. Data collection and analysis was performed using Bio-Plex Manager 4.1 software.

#### *2.7. Tube Formation Assay Using Matrigel Matrix*

Human Umbilical Vein Endothelial Cells (HUVEC) were isolated and cultivated according to the previously described protocol [43]. Wells in a chilled 96-well plate were coated with 50 μL of Matrigel matrix (with a reduced concentration of growth factors (Becton Dickinson, Franklin Lakes, NJ, USA)) and incubated at 37 ◦C for an hour. After the matrix had solidified, 1 × 104 endothelial cells were seeded in 100 μL onto the Matrigel surface by adding conditioned medium from the HEK293T cells previously transfected with various plasmid constructs. Non-transfected HEK293T medium and poor medium containing 10% FBS were used as a negative control. Complete medium containing 10% FBS and Endothelial Cell Growth Supplement (ECGS) 30 μg/mL (Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control. The formation of capillary-like structures was assessed after 6 h using phase-contrast microscopy. The total tube length and the number of formed branch nodes were calculated using the WimTube software package in the Wimamsis system (Available online: https://www.wimasis.com/en/WimTube, accessed on 19 March 2019)

#### *2.8. Statistical Analysis*

Each experiment was performed three times in replicates. All data are presented as mean with standard error of the mean (±s.e.). Multiple comparisons were tested by one-way analysis of variance (ANOVA) followed by a post-hoc Tukey test using the

GraphPad Prism 7 software (GraphPad Software, California, USA). The *p*-value *p* < 0.05 was considered statistically significant. Significant probability values were denoted as follows: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001, ns—no statistically significant difference.

#### **3. Results**

#### *3.1. Characterization of Multigenic Constructs Containing Picornavirus 2A-Peptide Sequences*

In the current study, recombinant plasmid vectors were designed, encoding codonoptimized sequences of VEGF and FGF2 genes and DsRed under the single control CMV promoter. In bi-cistronic (pVax1-VEGF-DsRed, pVax1-FGF2-DsRed) and tri-cistronic vectors (pVax1-VEGF-FGF2-DsRed) the Fu-2A-peptide sequence was incorporated between the target genes. All vectors were constructed based on widely used and clinically approved plasmid pVax1. The primary structure of DNA was verified via routine sequencing (data not shown). The quality of both the purified plasmids, and the presence of target inserts, were confirmed by restriction analysis. The resulting fragments corresponded to the expected molecular size (Figure 1).

**Figure 1.** Design and characterization of plasmid vectors. (**A**) Schematic of the genetic cassettes, containing therapeutic genes (Vascular Endothelial Growth Factor (VEGF165), Basic Fibroblast Growth Factor (FGF2)) and reporter fluorescent protein (Red fluorescent protein (DsRed)). (**B**) Schematic diagrams of the expression vectors. (**C**) Analysis of enzymatic digestion. Agarose gel electrophoresis. M—marker/HindIII (SM0103) and GeneRuler DNA LadderMix (SM0331).

#### *3.2. VEGF, FGF2 and DsRed Expression in Genetically Modified Cells In Vitro*

To confirm the expression of target genes, HEK293T cells were transfected with recombinant plasmid constructs. We found that the expression plasmids increase the expression of mRNA VEGF- and FGF2-modified cells compared with the empty vector (pVax1-Dsred) and non-transfected cells. Fluorescent microscopy analysis revealed DsRed

expression in all experimental groups. The immunofluorescent assay demonstrated a positive reaction for antibodies to VEGF and FGF2 in transfected cells (Figure 2A).

**Figure 2.** Expression of recombinant angiogenic factors in transfected HEK293T cells. (**A**) Immunofluorescent analysis of genetically modified HEK293T cells. Staining for VEGF (green) and FGF2 (red). Nuclei were counterstained using a DAPI solution (4 ,6-diamidino-2-phenylindole) (blue). Scale bar 100 μm. (**B**) mRNA expression of target genes (VEGF, FGF2) in HEK293T cells transfected with plasmids pVax1-VEGF-FGF2-Dsred, pVax1-VEGF-Dsred, pVax1-FGF2-Dsred, pVax1-Dsred. mRNA from cells was assayed by RT–PCR and quantified relative to 18S rRNA mRNA levels. mRNA expression in non-transfected cells (NTC) was considered as control. Data presented as average ± s.e.; *p* < 0.05 \* regarded as statistical significant differences (*n* = 3; \*\* *p* < 0.01; \*\*\*\* *p* < 0.0001 compared with control; ns—non-significant).

#### *3.3. Production of VEGF and FGF2 by Genetically Modified Cells*

The efficiency of VEGF and FGF2 secretion ex vivo was confirmed using indirect ELISA of cell lysates and supernatants collected from genetically modified cells. The ELISA results revealed statistically significant upregulation of VEGF secretion in both the supernatants and lysates of the cells modified with pVax1-VEGF-FGF2-DsRed (3629.68 ± 125.05 pg/mL). Increased VEGF production was also registered in supernatants of the cells transfected with pVax1-VEGF-DsRed (3530.00 ± 291.15 pg/mL) compared to non-transfected control (61.77 ± 3.03 pg/mL) (Figure 3A). Cells transfected with pVax1-VEGF-FGF2-DsRed (1396.00 ± 29.06 pg/mL) and pVax1-FGF2-DsRed (1728.00 ± 85.18 pg/mL) produced increased amounts of FGF2 compared to the cells modified with pVax1-VEGF-DsRed (16.73 ± 6.09 pg/mL) and in comparison to pVax1-DsRed and naïve cells as well (Figure 3B).

**Figure 3.** Analysis of VEGF and FGF2 concentrations in HEK293T cells transfected with obtained plasmid constructions. A total of 2.5 × 105 were seeded per well in a 12-well plate, and transfections were conducted after letting the cells adhere overnight. (**A**) VEGF concentration in supernatants and cell lysates of modified cells (pVax1-VEGF-FGF2-DsRed, pVax1-VEGF-DsRed, pVax1- FGF2-DsRed, pVax1-DsRed) and non-transfected controls (NTC); (**B**) FGF2 concentration in supernatants and cell lysates of modified cells (pVax1-VEGF-FGF2-DsRed, pVax1-VEGF-DsRed, pVax1- FGF2-DsRed, pVax1- DsRed) and non-transfected controls (NTC). Data presented as average ± s.e. of three independent repeats in independent samples, statistically significant differences \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.0001, \*\*\*\* *p* < 0.0001; ns—non-significant.

#### *3.4. Cytokine Profile Study of Genetically Modified Cells*

The effect of the upregulated expression on cytokine pattern was investigated by multiplex analysis of cell lysates and supernatants collected from genetically modified cells. A total of 27 proteins were analyzed and the expression of 15 proteins was detected. Non-transfected cells were shown to express soluble receptor (IL-1ra), pro-inflammatory (IFN-γ, IL-6, IL-7, IL-12(p70),), anti-inflammatory cytokines (IL-8, IL-10), chemokines (IP-10, MCP-1, RANTES), and growth factors (G-CSF, FGF2, GM-CSF, PDGF-bb, VEGF). Secretion levels of several cytokines were insignificantly higher in modified cells. Notably, in cell lysates and supernatants from pVax1-VEGF-FGF2-DsRed group, upregulated IL-1ra, IL-7, IL-8, IL-12(p70), IL-6, IL-10, IFN-γ, IP-10, RANTES, MCP-1, and growth factors G-CSF, GM-CSF, PDGF-bb were observed at levels 3–7 times higher compared to NTC. However, it was statistically not significant (*p* > 0.05). Increased production of the aforementioned factors was also observed in cells transfected with pVax1-VEGF-DsRed. Slight augmentation of IL-6, IL-8, MCP-1, IFN-γ, and PDGF-bb were registered in cell lysates in the pVax1- FGF2-DsRed group (*p* > 0.05). An expected augmentation of VEGF and FGF2 (*p* < 0.01) was scored in pVax1-VEGF-DsRed, pVax1-FGF2-DsRed, and pVax1-VEGF-FGF2-DsRed transfected cells (Figure 4) that coincided with the above-mentioned ELISA data (Figure 3).

**Figure 4.** Protein array analysis of cytokine, chemokine, and growth factor in genetically modified HEK293T cells (pg/mL). (**A**) Concentration of soluble molecules, secreted by transfected HEK293T cells. (**B**) Concentrations produced from factors in lysates from transfected HEK293T cells. Analytes was measured 48 h after cell transfection. Data presented as average (n = 4), asterisks (\*) indicate statistical significance compared with the control, as determined by one-way analysis of variance (ANOVA) followed by a post-hoc Tukey test (\*\* *p* < 0.01).

#### *3.5. Effect of Increased VEGF and FGF2 Expression on Capillary-Like Structures Formation by HUVEC In Vitro*

To evaluate the proangiogenic potency of plasmid constructs and the synergetic effect of secreted VEGF and FGF2, HUVEC cells were cultured on a three-dimensional Matrigel matrix supplemented with conditioned media from transfected cells (Figure 5). Conditioned media collected from cells transfected with pVax1-VEGF-FGF2-DsRed provoked the most significant stimulation on tube formation by HUVEC compared with cells supplemented with media obtained from pVax1-VEGF-DsRed, pVax1-FGF2-DsRed, pVax1- DsRed, and control HEK293T cells. Meanwhile, in the control groups (pVax1-DsRed and NTC), a few cellular structures that were not connected were observed. In contrast, other groups demonstrated a higher level of organization of capillary-like structures (tubule area and total length) and a higher level of tubular organization (number of loops and bifurcations). In the pVax1-VEGF-DsRed group the total length of formed tubes was less (166640.30 ± 1297.53px) compared to pVax1-VEGF-FGF2-DsRed (21409.00 ± 2183.81px). However, in the pVax1-FGF2-DsRed group (20150.30 ± 1289.05px), the total length of the formed tubes was not significantly different from the pVax1-VEGF-FGF2-DsRed group21409.00 ± 2183.81px) (Figure 5B). Similar results were obtained concerning the number of branch points discovered; in the pVax1-VEGF-FGF2-DsRed (190 ± 28.86), pVax1-VEGF-DsRed (139.00 ± 15.30), and pVax1-FGF2-DsRed (187.66 ± 16.259), the numbers were higher in comparison to the control groups pVax1-DsRed (86.66 ±15.45) and NTC (70.00 ± 3.7) (Figure 5C). Thus, augmented expression of VEGF and FGF2 contributed to the formation of a denser network of capillary structures compared to other groups, resulting in increased amount of loops forms, with pVax1-VEGF-FGF2-DsRed (71.5 ± 11.61), pVax1-VEGF-DsRed (44.25 ± 6.00), and pVax1-FGF2-DsRed (69.66 ± 11002) showing higher numbers than control groups pVax1-DsRed (20.33 ± 7.12) and NTC (11.33 ± 2.18)

(Figure 5D). In the pVax1-VEGF-FGF2-DsRed and pVax1-FGF2-DsRed groups, the percentage of cell coverage increased by 10% compared to control groups pVax1-DsRed and NTC. This up-regulation was statistically significant and correlated with other indexes, including the differential number of loops, branch points, and total tube length (Figure 5E).

**Figure 5.** Analysis of tube formation by human umbilical vein endothelial cells (HUVEC). A total of 1 × 104 HUVEC were seeded per well in a 96-well plate. HUVECs were cultivated with conditioned media collected from HEK293T cells transfected with pVax1-VEGF-FGF2-DsRed, pVax1-VEGF-DsRed, pVax1-FGF2-DsRed, pVax1-DsRed, and non-transfected control as well (NTC). ECGS complete medium containing 10% FBS and Endothelial Cell Growth Supplement (30 μg/mL). Medium+ 10 FBS –medium containing 10% FBS without ECGS. (**A**) Brightfield microscopy image, scale bar 200 μm. (**B**) Length of formed tubes (measured in px), (**C**) Number of branch points formed, (**D**) Number of loops, (**E**) Area of cell coverage. Analysis was carried using Wimasis imaging software package. Data presented as average ± s.e. \* indicates statistically significant data relative to control (n = 3; \*\* *p* < 0.01; \*\*\* *p* < 0.0001 ns—non-significant).

#### **4. Discussion**

We designed and tested multigene vectors encoding the FGF2, VEGF, and DsRed genes, or combinations of these, combined through the picornoviral 2A-peptide sequences. Our data demonstrated that the expression of mRNA and FGF2 and VEGF proteins in vitro

was higher than that observed in the control groups (cells modified with the DsRed plasmid and non-modified cells).

The selection of an appropriate vector system is believed to be one of the essential aspects of developing gene therapy drugs. The vector pVax1 is a non-immunogenic optimized expression system. The content of eukaryotic DNA sequences is critically reduced, thus minimizing the vector's chromosomal integration into the host cell genome. Besides this, pVax1 has been approved by the American Food and Drug Administration (FDA) and is currently used widely to develop DNA vaccines [64]. Considering the world's current situation due to the global spread of severe acute respiratory syndrome 2 (SARS-CoV-2), DNA vaccine development remains an urgent issue [65]. In particular, multicistronic recombinant constructs could be used for the simultaneous equimolar expression of several immunogenic proteins of differing pathogens.

Gene co-expression systems make it feasible to increase the induction efficiency of therapeutic angiogenesis due to the simultaneous delivery of several proangiogenic factors. Application of 2A-peptides may become a strategy employed to achieve stable gene co-expression [66]. This approach is attractive in the expression systems' design when encoding combinations of therapeutic molecules in the same transcriptional unit and provides a large packaging capacity [67]. In addition, it has been tested in a wide range of eukaryotic cells [68,69]. High transgene expression has been confirmed, as has good cleavage of synthesized recombinant proteins [50,68], and the absence of immune responses to system components has also been verified [68]. The application of multicistronic expression systems will therefore simplify the development procedures needed for polygenic constructs to achieve the simultaneous delivery of several therapeutic genes to a particular cell [70,71]. In the current study, HEK293T cells were used as a model system to characterize recombinant plasmids and evaluate the effect of over-expression of VEGF and/or FGF2 on the secretion profile of genetically modified cells. The possibility of successful modulation of the secretion profile of HEK293 by the action of various external factors has been demonstrated previously. In particular, co-cultivation of HEK293 with mesenchymal stem cells that expressed lipocalin 2 resulted in an increased expression of growth factors HGF, IGF-1, and FGF2 by HEK293 cells [72]. Confirmed changes in the secretion of HEK293 IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, IFN-γ, TNF-α due to the influence of bovine serum albumin have also been published [73].

In our study, multiplex analysis of soluble factors in the supernatants and cell lysates of HEK293T revealed augmentation of several cytokines, chemokines, and growth factors in transfected cells. However, the differences found, except for VEGF and FGF2, were not statistically significant. Therefore, we suggest that for a deeper understanding of the functioning and effects of VEGF and FGF2 overexpression on the state of modified cells, it is necessary to expand the range of analytes investigated, as well as to select cells that have a pronounced autocrine and paracrine potential relating to the production of the recombinant factors used. In our opinion, mesenchymal stem cells from various origins could serve as appropriate candidates. In particular, we have previously demonstrated that genetic modification and overexpression of proangiogenic factors by cells can modulate the secretory profile of stem cells from human adipose tissue [74]. Shanshan Jin et al., previously reported that overexpression of FGF2 by human gingival mesenchymal stem cells enhanced their secretion of VEGF and TNF-β [75]. Similar results were obtained by Yukita et al., where recombinant FGF2 increased the expression of glial neurotrophic growth factor (GDNF) by dental follicle cells [76]. We assume that such adaptations of the secretome to overexpress angiogenic factors might further increase the transplanted cells' therapeutic potential. The current assay has shown a high correlation between the various techniques used to verify the created structures' functionality. Using an in vitro model of angiogenesis, we have shown that the conditioned medium collected from genetically modified cells caused a stimulating effect on capillary formation by endothelial cells. The presented data are consistent with our earlier obtained results studying two-cassette plasmid vectors providing independent expression of two growth factors [43]. The results

indicate that, in the studied model, FGF2 more effectively affects the formation of capillarylike structures by human endothelial cells as compared to VEGF. The slight effect of secreted VEGF on the formation of vascular-like structures in the present study is likely to be due to the short exposure period. When using recombinant VEGF or VEGF expressing plasmid constructs, several studies have demonstrated that significant induction of angiogenesis is only observed 20–30 days after therapeutic exposure [77,78]. At the same time, significant differences were not observed between the pVax1-VEGF-FGF2-DsRed and pVax1-FGF2 groups in the present study. We assume that the critical contribution to the formation of vascular-like structures in vitro is mediated by FGF2 expression. These results are consistent with previous reports showing that FGF2 induced the highest blood vessels density in a mouse cornea model [79]. Cartland et al. have previously indicated that FGF2 is more effective at stimulating angiogenesis in vitro and in vivo than VEGF and TRAIL [80]. This phenomenon may be because VEGF and FGF2 act on a wide range of receptors and, accordingly, activate various signaling cascades and stages of angiogenesis [79–81].

It is worth emphasizing that pro-angiogenic factors are active even in picomolar concentrations and, as a consequence, a slight local increase in their concentration is sufficient to achieve a therapeutic effect. Moreover, in different living species, presumably, since trophic factors are evolutionarily conserved molecules, the same factors can activate similar biological effects. In this regard, it is therefore no coincidence that human molecules, such as growth factors, might be active in various model organisms. Moreover, codon optimization is expected to preserve the biological potential of the synthesized molecules [82]. Simultaneously, alternative data are accumulating, and indicating the negative effect of codon optimization on the translation and structure of the synthesized protein [83], which introduces one more variable that must be taken into account when creating effective, optimized gene therapy systems. Our results show that recombinant constructs expressing different genes are putatively implementing discrete patterns of angiogenesis. Their efficacy for therapeutic use in the induction of angiogenesis should be tested using in vivo models.

#### **5. Conclusions**

Our work has constructed and tested, in vitro, a multicistronic system that ensures simultaneous efficient delivery of several therapeutic growth factor genes into cells. Data on the functionality of the synthesized recombinant proteins as the part of the multicistronic construct have also been obtained. We have demonstrated that the proangiogenic factors synthesized in the secretome of genetically modified cells exhibit stimulating effects on the formation of capillary-like structures by HUVEC in vitro. This approach can be used to develop and test gene therapy protocols for various human diseases that require the simultaneous expression of several transgenes to stimulate therapeutic angiogenesis and other regenerative processes.

**Author Contributions:** Conceptualization, I.I.S., V.V.S. and A.A.R.; methodology, A.P.K. and A.A.R.; investigation, D.Z.G., E.E.G., G.E.S., A.F.M., M.N.Z.; validation, I.I.S. and D.Z.G.; writing–original draft preparation, D.Z.G. and I.I.S.; writing—review and editing, I.I.S. and C.S.R.; supervision, A.A.R. and A.P.K.; funding acquisition, A.A.R. and I.I.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially supported by the Russian Foundation for Basic Research grant No 18-44-160029 and by a subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities (project 0671-2020-0058).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.

**Acknowledgments:** This work is part of Kazan Federal University Strategic Academic Leadership Program.

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

#### **References**


**1. Introduction**

Determining the beginning and end of an eruption, and forecasting what will be the dominant eruption style, are equally important objectives, crucial for hazard assessment.

Satellite sensors have been increasingly employed for operational monitoring of volcanic thermal features as for example the geostationary Spinning Enhanced Visible and InfraRed Imager (SEVIRI) instrument on board the geostationary Meteosat Second Generation (MSG) satellite [1–4] and the Geostationary Operational Environmental Satellites (GOES) [5], or the polar Moderate Resolution Imaging Spectroradiometer (MODIS) [6], the Along Track Scanning Radiometer (ATSR) [7] and the advanced very-high-resolution radiometer (AVHRR) [3,8–12], on board respectively of the NASA Terra/Aqua, the ESA-ENVISAT, and the NOAA satellites.

In spite of its coarse spatial resolution, the high temporal rate of the geostationary systems represents an important tool for volcano monitoring (e.g., [13–15]). In particular, SEVIRI has 12 spectral channels from visible to thermal infrared, a spatial resolution of 3 km at nadir and a high temporal resolution that ranges from 15 min (earth full disk (EFD)) to 5 min (rapid scan mode (RSM) over Europe and Northern Africa). Exploiting these characteristics, SEVIRI allows a precise timing of the early phase of an eruption and almost a continuous monitoring of volcanic activity from the source to the atmosphere [4,16]. The SEVIRI data are collected in real time from a Meteosat-8 ground station antenna operating from 2010 at Istituto Nazionale di Geofisica e Vulcanologia (INGV) in Rome (Italy) [17,18].

In this work, the SEVIRI measurements have been used to investigate the eruptive processes and to discriminate different styles of volcanic activity by applying a novel procedure based on the correlation between the radiance growing rate at the beginning of the eruption with the specific type of volcanic activity. As test case, the 2013, 2015 and 2017 Etna events have been considered, and the results of the remote sensing analyses have been validated using ground-based information and literature references.

The paper is organized as follows: Section 2 outlines eruptions on Mt. Etna in the 2011–2015 period while Section 3 describes the method developed for the discrimination of the different styles of volcanic activities. In Sections 4 and 5 the results are presented and discussed, respectively. Final conclusions are drawn in Section 6.

#### **2. The 2011–2015 and 2017 Etna Activities**

In the 2011–2015 period, the eruptive style of Mt. Etna changed from predominantly effusive to more explosive activity. Strombolian events became more frequent and intense and were often associated with strong explosions, ash emissions, and lava fountaining paroxysmal episodes (e.g., [16]. The duration of these "paroxysmal" episodes varies from minutes to hours [4,19–22]. Between 2 and 8 December 2015, all four summit craters of Mt. Etna generated an extraordinary sequence of eruptive events. The activity consisted of high eruption columns, Strombolian explosions, lava flows, and widespread ash falls [23,24].

In February 2017, a vigorous strombolian activity started from the South-East (SE) crater producing a small lava flow. The direction of the lava flow was towards the southwest flank and flowed down slowly to an elevation of about 2850 m asl. After having lasted approximatively 48 h, the intense explosive-effusive activity began to weaken quickly. On 15 March new lava flows erupted from a fissure vent at the southern base of the SE crater complex. These lava flows descended into the *Valle del Bove* at several locations on the rim between 2500–2700 m elevation. On their descend down the steep western headwall, the lava flow fronts suffered partial collapses and interacted explosively with snow, both leading to the generation of small pyroclastic density currents that produced tall brown ash plumes [25].

#### **3. Materials and Methodology**

Statistical analysis was performed for Etnean eruptions using the 3.9 μm SEVIRI channel (Medium InfraRed-MIR channel) acquired from 2011 to 2017. Most of these eruptions were paroxysmal events, including Strombolian explosive activity, often associated with ash/gas emissions, and lava fountaining. Unfortunately, the statistics on lava flows are reduced to two episodes: the two-day long lava flow in December 2015 and the intense effusive activity that occurred from February to March 2017. The analysis of different eruptions has highlighted different radiance trends at the early stages of eruptions.

The December 2015 Etna eruption was characterized by the succession of several eruptive styles in a matter of days. Figure 1 shows the SEVIRI MIR radiance time series, measured from a single SEVIRI pixel centered over the Mt. Etna summit area and collected from 2 to 8 December 2015. The most powerful episode occurred between 2 and 3 December 2015 from the Voragine crater (Ep 1) when high lava fountains were produced without lava flows emitted. The MIR radiance drop that suddenly appeared in the MIR signal on 3 December, clearly indicates the absorption due to the ash emitted during the event [4]. From 4 to 5 December further powerful episodes occurred at the Voragine with strong emission of ash and gases (Ep 2). In this case, the scene was extremely cloudy and the radiances appear low compared to Ep 1 and its highly variable trend [4]. Finally, the activity shifted to the New Southeast Crater on 6 December 2015 (Ep 3), where Strombolian activity and lava flow emission lasted for two days and were fed by the most primitive magma of the study period, that is magma that underwent minimal igneous differentiation from the original composition [24]. Together with the MIR radiance trend, in Figure 1 are also drawn the growing rates for both the lava fountain (red dashed curve) and the lava flow (blue dashed curve). The trend of the 2 and 3 December lava fountain shows a gradual rise to a peak in about 24 h with radiance growing at an exponential rate. The maximum

intensity lasts less than one hour, after which the radiance quickly starts to decrease at an exponential rate. From 4 to 5 December, two further potent episodes occurred at the Voragine, characterized by ash emissions and weak Strombolian activity. Radiance trend shows a high variability with the presence of peaks in correspondence of paroxysmal episodes. However, the thick cloud coverage present on those days significantly influences the signal detected at the sensor. Therefore, it is not reliable to characterize this activity based on radiance changes [4]. The trend of the 6–8 December lava flow suddenly changes from steady to rising and the maximum radiance is reached after a few minutes from the beginning of the eruption. Then the radiance is maintained at the maximum value for the duration of the emissions.

**Figure 1.** Spinning Enhanced Visible and InfraRed Imager (SEVIRI) Mid-Infrared (MIR) radiance acquired during the 2–8 December 2015 Mt. Etna eruption. Three different eruptive episodes occurred: lava fountaining (**Ep 1**), ash emission (**Ep 2**), and lava flow **(Ep 3**). Different episodes clearly show different trends that can be recognized a posteriori. However, differences are detectable even at the early stages of the eruption. An exponential trend and a sub-vertical trend seem to best fit the initial radiance increment for Ep1 (**red dashed line**) and Ep3 (**blue dashed line**) respectively.

The qualitative analysis of these three distinct episodes clearly shows strong differences in the radiance trends. It is quite easy to distinguish the three episodes knowing the overall trend retrospectively. However, for prevention reasons, it would be important to recognize the type of volcanic activity at the earliest stages of the eruption.

The basic idea is that when an eruption begins, the SEVIRI MIR radiance increases as a function of the temperature and the volume rate of the erupted products. There are two mechanisms that mainly contribute to defining the speed at which the integrated pixel temperature increases. On the one hand, the type of volcanic products generated by the eruption and their eruptive rates. For example, mixtures of ash, lava blebs and broiling gas at Mt. Etna are normally at temperatures between 200 ◦C and 700 ◦C, but they can exceed temperatures of 1000 ◦C, while the measured eruptive temperatures of Etnean lava flows range between 1080 and 1095 ◦C [26]. On the other hand, products such as clouds, volcanic

ashes, and plumes can act as a shield to radiation and partially or completely absorb the radiance emitted to space [27]. Both mechanisms affect the MIR radiance measured by the sensor, therefore, its trend has the potential to discriminate between eruptive styles. Herein, we aim to correlate the radiance growing rate at the beginning of the eruption (ramp) to the specific type of volcanic activity. By exploiting the SEVIRI RSM resolution a time-window of radiance values has been considered and analyzed in terms of signal processing. Changes in "signal" are detected at a certain time (t0) using a moving window of fixed width, which is incremented at every SEVIRI acquisition.

Signal analysis is performed on SEVIRI MIR data in order to obtain optimal smoothing of radiance time-series. The smoothing removes noise introduced by radiometric calibration and atmospheric effects caused by passing of transitional clouds in the investigated scene. The literature on time-series smoothing and denoising techniques is vast and encompasses different methods. Among the most renown and utilized methods are the Savitzky-Golay and Hodrick-Prescott filters, the Hilbert-Huang transform (HHT), the ample class of kernel filters, as well as wavelet analysis. Herein, we propose a method that uses the wavelet transform [28] to analyze the SEVIRI time-series.

#### *3.1. Wavelet Transform: the 'à trous' Algorithm*

The *à trous* algorithm of discrete wavelet transform is a powerful tool for multiscale (multiresolution) analysis of images [28]. The *à trous* algorithm allows performing a hierarchical decomposition of an image into a series of *scale layers*, also known as *wavelet planes* [29]. Each layer contains only structures within a given range of characteristic *dimensional scales* in the space of a *scaling function*. The decomposition is done throughout a number of *detail layers* defined at growing characteristic scales, plus a final *residual layer* or *smoothing layer*, which contains the rest of unresolved structures. By isolating significant image structures within specific detail layers, detail enhancement can be carried out with high accuracy. Similarly, if noise occurs at some specific dimensional scales in the image, as is usual in most cases, by isolating it into appropriate detail layers we can reduce or remove it without affecting significant structures. A complete description of the implementation of this algorithm can be found in Bijaoui and Giudicelli [28]. Herein, the *à trous* algorithm has been adapted to remove the effect of background noise and to isolate changes due to volcanic activity in the moving time-window.

Plate 2a of Figure 2 shows a time-series of MIR radiance obtained for a short lava fountain event occurred at Mt. Etna from 13 to 15 December 2013. The detail layers and the residual layer from wavelet transform are shown in Plates 2b–e. Both detail and residual layers have potentials to be used for the assessment of eruptive phenomena. High-frequency changes occur at the earliest decomposition steps. Therefore, detail coefficients of the first scale layer are ideal to trigger the initial phase of the eruption (Plate 2b). One property of the wavelet transform is to have a sampling step proportional to the scale [28]. This implies that significant structures (e.g., local maxima) in lower-detailed scale layers match structures in higher-detailed scale layers (Plates 2b, c, d). This property allows "following" the eruptive signal in subsequent transformations where the higher frequency content of the signal is progressively eliminated.

**Figure 2.** An example of multiscale decomposition with the *à trous* discrete wavelet transform algorithm. Seven detail layers have been generated with dyadic scaling sequence (only three are shown for clarity, **Plates 2b**, **2c,** and **2d**). Detail layers are generated for a growing scaling sequence of powers of two. The layers are generated for scales of 1, 2, 4, 8, ... samples. For example, the fourth layer contains structures with characteristic scales between five and eight samples. Wavelet decomposition results in a border distortion which increases with the power of two at each decomposition scale (indicated in figures with grey areas). The original SEVIRI MIR radiance time-series is shown in **Plate 2a**. Detail layers at increasing characteristic scales contain larger signal structures. At the end of the sequence is the residual layer (**Plate 2e**), which contains all of the remaining unresolved structures.

**Figure 3.** SEVIRI MIR radiance acquired during December 2015 Mt. Etna eruption. Red line is the smoothed layer derived from the *à trous* discrete wavelet transform. The smoothed layer allows for analysis of the overall trend without high-frequency noise due to instrumental noise, transient weather effects, and acquisition failures.

#### *3.2. Statistical Analysis*

The *smoothing layer* of the discrete wavelet transform allows capturing important patterns in the radiance data, while leaving out noise or other fine-scale structures/rapid phenomena. Herein, we propose a statistical approach to characterize the variability of the dataset in the *smoothing layer*.

Useful statistics for measuring the variability of a data set are skewness, kurtosis, and gradient. Skewness and kurtosis statistics can help us to assess certain kinds of deviations from normality of our data-generating process. Skewness is a measure of symmetry, or more precisely, the lack of symmetry. A distribution, or data set, is symmetric if it looks the same to the left and right of the center point. Skewness determines whether a distribution is symmetric about its maximum.

Kurtosis is defined as the degree to which a statistical frequency curve is peaked. Kurtosis is a measure of whether the data are heavy-tailed or light-tailed relative to a normal distribution. That is, data sets with high kurtosis tend to have heavy tails or outliers. Data sets with low kurtosis tend to have light tails or lack outliers. Finally, the gradient is defined as the slope of the line that interpolates the dataset of the moving window. The gradient measures the rate of change of radiance with time.

Time series analysis of SEVIRI data from 2011 to 2017 showed that kurtosis and gradient are most appropriate to characterize the relationship between radiance variations and eruptive styles.

#### **4. Results**

The sensitivities of the results were tested against different trends. Figure 4 shows the kurtosis (black line) and gradient (blue filled curve) values derived from *smoothing layer* (red line) at every SEVIRI acquisition (every 5 min) during the 2–8 December 2015 Etna eruption.

The kurtosis coefficient is often regarded as a measure of the tail heaviness of a distribution relative to that of the normal distribution. However, it also measures how much "peaked" a distribution is. When interpreting kurtosis, the normal distribution is used as a reference. A positive kurtosis implies a distribution with more extreme possible data values (outliers) than a normal distribution, thus fatter tails (*Leptokurtic distributions*). A negative kurtosis implies a distribution with less extreme possible data values than a normal distribution, thus thinner tails (*Platykurtic distributions*). Finally, distributions with zero kurtosis have roughly the same outlier character as a normal distribution (*Mesokurtic distributions*).

**Figure 4.** MIR radiance acquired during the December 2015 Etna eruption. The kurtosis (**black line**) and the gradient (**blue filled curve**) trends were derived from the *smoothing layer* (**red line**) at every Meteosat Second Generation (MSG) acquisition (every 5 min). Highest values of kurtosis (> 0) and gradient (> 1.5) occur at the beginning of the lava flow episode (6 December).

The ratio of kurtosis to its standard error can be used as a test of normality (that is, you can reject normality if the ratio is less than −2 or greater than +2). A large positive value for kurtosis indicates that the tails of the distribution are longer than those of a normal distribution. A negative value for kurtosis indicates shorter tails (becoming like those of a box-shaped uniform distribution).

Normalized kurtosis shows that the spread of the observations was slightly less than what would have been expected under the normal distribution. Kurtosis is less than −1.3 during non-eruptive phases and when the eruption reaches the highest point of intensity measurable with SEVIRI data. The normalized kurtosis trend indicates that outliers mainly occur at the beginning and the end of the eruptive episodes. Therefore, outliers in the kurtosis distribution can be used for hot-spot detection to identify the initial and final phases of the eruption. The magnitude of the outliers appears to be different depending on the eruptive episode. The magnitudes of the initial outliers are −1.4, −0.1, and 0.45 for the lava fountain, the explosive activity, and the lava flow episode, respectively.

The gradient (slope) of the line that best fits the *smoothing layer* distribution indicates the steepness of the *moving window* distribution at every SEVIRI acquisition. Positive slope indicates increments in radiance and, therefore, possible increases in volcanic activity. Again, the maximum slope at the onset of each eruptive episode appears to vary depending on the type of volcanic activity. A slope magnitude of 1.0, 0.4, and 1.7 is obtained at the onset peak for the three episodes.

Figure 5 shows the *smoothing layer* (red line), the normalized kurtosis (black line) and slope (blue filled curve) for the 28 February and the 15 March eruptive events. The two events, both characterized by the presence of lava flows, show a relatively high value of gradient and kurtosis at the onset of the eruption. The lava flow generated by the 28 February eruption displays a gradient of 2.1 and normalized kurtosis of 2.0. The lava flow of 15 March, much stronger and longer than the previous episode, shows maximum gradient and kurtosis values of 2.5 and 4.4, respectively.

**Figure 5.** The *smoothing layer* (**red line**), the normalized kurtosis (**black line**) and slope (**blue filled curve**) for the 28 February and 15 March eruptive events. The lava flow generated by the 28 February eruption displays a gradient of 2.1 and normalized kurtosis of 2.0 at the onset of the eruption. The lava flow of 16 March, much stronger and longer than the previous episode, shows maximum gradient and kurtosis values of 2.5 and 4.4 respectively.

Threshold values were calculated using an eight-hour (100 samples) temporal window. The window width was experimentally chosen to optimize the signal-to-noise ratio of the radiance wavelet transform. A larger window implies that signal is smoothed over a longer period which results in losing details. A narrow window is more sensitive to high-frequency noise and, therefore, susceptible to false positives [30]. Figure 6 shows the *smoothing layer* (red line), the normalized kurtosis (black line) and slope (blue filled curve) for the 14 and 15 December 2013 Etna lava fountain. Vigorous Strombolian activity with associated ash plumes was observed at the New Southeast Crater (NSEC) (Volcano Discovery, Etna volcano activity updates: December 2013). The activity generated a small lava flow (probably mainly fed by rapid accumulation of liquid spatter) moving towards the SE flank with the presence of exploding magma bubbles towards the end of the eruption [31]. Under a statistical point of view, this paroxysmal event is very similar to the 3–5 December paroxysm (Ep 1) with kurtosis outlier of 0.05 and a maximum gradient of 0.45. Both episodes were characterized by Strombolian activity, intense ash emissions, and small effusive activity. Many of the paroxysms that took place during the investigation period fall into this type of scenario.

The transitional phases from Strombolian activity to lava fountaining can be observed and detected. On the evening of 16 March 2013, NSEC produced an intense episode of lava fountaining. This event, one of the most violent of the 2013 series of paroxysms, was preceded by a long "prelude" of Strombolian activity [32] that started on the afternoon of 15 March and was followed by weak, discontinuous activity at the Voragine [31]. The transition from the strombolian to the lava fountaining episodes is recognizable in the slope (blue filled curve) trends of Figure 7. It is clear from the plots that we have an increase in the slope magnitude from 0.5 to 1.5 when the lava fountaining begins and the lava starts to overflow through the deep breach in the southeastern rim of the NSEC [33].

**Figure 6.** Statistics for the 14–15 December 2013 Etna lava fountain. Relatively low values of kurtosis (<0) and gradient (<0.5) characterize this kind of activity at the onset of the eruption.

**Figure 7.** The analysis of statistical indexes allows highlighting transitions between different eruptive phases within the same activity. The transition from the Strombolian phase to the lava fountaining is well recognizable when we look at the peak (**blue arrow**) in gradient values (**blue filled curve**) during the 16 March 2013 Etna eruption.

#### **5. Discussion**

Our results suggest a relationship between the rate of increment in radiance (and thus temperature) and the nature of the volcanic process (i.e., eruptive styles as Strombolian activity, lava fountains and lava flow) causing that increment. Statistical analysis of SEVIRI MIR radiance trends highlighted the involvement of at least two different heating mechanisms for eruptions at Mt. Etna.

The first mechanism involves rapid increases in radiance and appears to be associated with effusive activity. Statistic kurtosis and gradient provide a measure of the steepness of the radiance trend. Relatively high values of kurtosis and gradient are mainly associated with the presence of active lava flows. In this case, Kurtosis and gradient magnitudes vary in the range of 0.0–5.0 and 0.5–3.0 respectively. We observed that statistics for small flows, as those generated by lava fountaining lava spatter or intra-crater lava ponding, show lower values of kurtosis (−0.5–0.5) and gradient (0.5–1) compared with values associated with long-term lava flows (see 2013 March case study of Figure 5).

The second mechanism is characterized by slope values usually less than 0.5 and refers to Strombolian events, often associated with paroxysmal activity and ash plumes. Moreover, the kurtosis outlier values remain below zero.

To understand and interpret the differences in radiance rates at the beginning of an eruption is not an easy task. However, we believe that such differences are the results of differences in eruptive and depositional processes of the different hot volcanic products (i.e., lava, ash, volcanic bombs). Eruption style controls both surface heating, the function of the output rate of lava and/or pyroclasts at a different temperature, and surface cooling, the function of the nature, temperature, and dispersal of the products. These heating and cooling mechanisms may be acting at the same time and thus explain the observed radiance behavior.

Temperatures of erupting magmas at Mount Etna are shown to be determined within a few ◦C in the range 1000–1200 ◦C [26]. The magmatic temperatures and petrological characters appear closely related to the volcanic activity. The alkaline basic magma of Mount Etna extrudes as lava at a nearly constant temperature of 1080 ◦C, but during explosive paroxysmal eruptions, the magma temperature is higher (1125 ◦C and possibly more). However, the small fragments of ash, lapilli, and bombs erupted during paroxysmal eruption fly through the air and cool quickly along their path through the atmosphere. Therefore, pyroclasts ejected in the atmosphere during explosive activity cool much faster than lava flows originated at effusive vents. In addition, gases and ash particles thrown into the atmosphere during volcanic eruptions influence the measured radiance. The particles spewed from volcanoes have the ability to absorb the radiation emitted by erupted materials shading the radiance signal detected at the sensor.

An important role is also played by the volume of the volcanic deposits generated during the eruption. The fraction of the pixel occupied by "hot products" depends on the type of volcanic eruptions and their spatial distribution. For example, explosive eruptions at Mt. Etna are mainly concentrated at the summit craters while lava flows tend to expand and occupy an increasingly larger area as the eruption continues. Therefore, the radiance detected at the sensor is a function of the temperature of the erupted materials and the fractional area occupied by those materials.

Validation was conducted by comparing our results with literature data using a time-series of SEVIRI data from 2011–2017 (4). We used threshold values of 0.1 and 0.5 for the kurtosis and the slope, respectively, to distinguish between effusive and explosive eruptions in the first 8, 16, and 24 h from the onset of the eruption. We found that, 79% of the eruptions were correctly interpreted in the first eight hours, 85% in the first 16 h, and 93% after a day from the beginning of the eruption.

However, several issues need to be taken into account. First, information on eruptions derived from monitoring bulletins is mainly qualitative information. Therefore, it is difficult to accurately determine the intensity of the eruptions and their duration from available literature data. Second, literature data are mainly referring to thermal camera data, remote sensing and field observations. These observations are all weather dependent. Third, our time-series suffers from data loss issues due to acquisition failure. Data loss and noisy images are suitable to generate artifacts that lead to

the presence of a false outlier. Finally, events occurring between 2011 and 2017 are mainly short-term paroxysmal eruptions, while long-term effusive eruptions are scarce.

The transition from Strombolian to fountaining activity is well documented [19,32]. Since 1999, Mount Etna has been characterized by episodic lava fountaining, each episode identified by initial Strombolian activity followed by transition to sustained fountaining to feed high effusion rate lava flow [32]. Such transitional behavior introduces further uncertainties into the identification and classification of the erupting style.

Mt. Etna, with its dense, multiparametric array of monitoring networks, provided an ideal test case for our analysis. Future integration of the proposed method will complement the existing networks and provide an additional monitoring tool independent from ground support. Finally, our results represent a promising step toward similar application to less monitored volcanoes, also in remote areas. Such application will require the threshold values empirically calculated for Mt. Etna to be adapted to specific cases or, alternatively, to be derived by ad-hoc models of eruption-dependent thermal radiation.

#### **6. Conclusions**

Eruption monitoring by satellite remote sensing still requires validation and refined, ground-tested methodologies. In this study, we explored the capability of high temporal resolution satellite data to discriminate and, to a limited extent, predict erupting styles at Etna volcano. A new statistical approach based on the wavelet transform of time-series of SEVIRI radiance data acquired at 5–15 min acquisition rate in the MIR spectral band (3.9 μm) is presented. Statistical analysis of processed data show potential in predicting eruptive styles. Statistic kurtosis and gradient derived from the analyzed time series allowed to recognize between two different eruptive styles: 1) paroxysmal events, characterized by Strombolian explosive activity, often associated with ash emissions, and lava fountaining (15–17/03/2013, 13–17/12/2013) and 2) lava flows (2–8/12/2015 and 26/02/2017–21/03/2017). It must be stressed that our methodology applies to data acquired at the onset of the eruption in order to give a quick response in terms of alert. The results obtained by this method have a considerable degree of trustworthiness in the first eight hours from the beginning of the eruption.

By considering the reliability of the proposed approach, the authors believe that it can be also applied worldwide, in particular for volcanoes lying in remote areas.

**Author Contributions:** V.L. conceived the idea, analyzed data, implemented wavelet transform, and wrote the manuscript with contributions from all authors. M.M. and M.S. processed satellite data and exported time-series for wavelet transform. S.C. and J.T. carried out the study of paroxysmal events with particular emphasis on the effects of gaseous volcanic emissions, ash plumes and explosive episodes. All authors contributed to the ideas, writing, and discussion.

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

**Acknowledgments:** We thank the anonymous reviewers for their careful reading of our manuscript and their many insightful comments and suggestions.

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

#### **References**

© 2019 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 (http://creativecommons.org/licenses/by/4.0/).


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**Figure 6.** *(a) Tongue defect after resection, (b) RFFF harvest and (c) RFFF insetting.*

**Figure 7.** *(a) ALT flap design and (b) ALT flap harvest.*

primary and salvage surgery (**Figure 8**). This flap based on the thoracoacromial artery can be raised as a myocutaneous or fasciocutaneous flap. It is reliable, robust, and easily harvested in terms to tongue reconstruction and can provide muscle and skin to fulfill the tongue and floor of the mouth and effectively separate the oral cavity from the neck. It must be suspended across the mandibular arch by either suturing

**151**

**4.3 Cheek**

**Figure 8.**

*Major pectoral flap harvest.*

*Modalities and State of Art in Oral Cancer Reconstruction*

time but are an excellent alternative when needed.

The cheek resection is done less frequently except in some countries like India, where cheek cancer is frequent and as a consequence of chewing tobacco; usually its oncological resections leave a complex defect that includes skin and mucosa in an area where a functional lip is required to avoid food spillage. The consequent

to the pterygoid musculature or securing to the mandible using drill holes to avoid and prevent the flap from falling [26]. This flap is considered a horse battle in rescue setting when a free flap fails. When the defect includes mandible, during the reconstruction it must have keep in mind that mandible contributes to airway stability, oral competence, speech, deglutition and mastication, so the goal of this reconstruction must include the preservation of the ability to open the mouth, occlusion, and the restoration of the inter arch continuity solutions to promote dental implants and restore chewing as mentioned in floor of mouth defects extended to mandibula. Not reconstructing the central defects will conclude in loss of the lip support with Andy Gump deformity, and not reconstructing the lateral defects will cause malocclusion and lateral shift in the position of mandible, so any intent must be done to reconstruct the mandible. Options in reconstruction include metal plates (**Figure 7**), non-vascularized bone grafts, osteomyocutaneous pedicled flaps, and osteocutaneous free flaps. Fixing soft tissues just with plates was widely used in the past and usually results in extrusion intraorally, external exposure or fracture of the plate up to 60% of the cases with a worst defect and a very poor functional outcome [27]. Autogenous bone grafts from iliac crest, scapula, or calvarium usually end in no vascularization of the new bone and its atrophy even more if radiation is added to the treatment, and finally similar results as the plating alone are obtained, so similarly they are no more used. Currently the gold standard in mandible reconstruction is the osteocutaneous free flaps (**Figure 9a**–**c**) and carries the same consideration as mentioned in floor of mouth reconstruction with a trend to perform a first time micro vascularized bone reconstruction with dental implants mainly in a previous dentulous young patient [28]. In an aged edentulous patient in the reconstruction setting, there is most likely no need to be aware for dental implants unlike dentulous young patient. Again, in selected patients with poor clinical condition and not suitable for a long procedure, a osteocutaneous pediculate flap such as a osteomyocutaneous trapezius flap [29] or a bicortical parietal osteofascial pedicled flap [30] can be perform providing a better functional result compared with just soft tissue coverage. Both flaps require experience, skills, and anatomic knowledge to harvest them in a short period of

*DOI: http://dx.doi.org/10.5772/intechopen.91049*

*Modalities and State of Art in Oral Cancer Reconstruction DOI: http://dx.doi.org/10.5772/intechopen.91049*

**Figure 8.** *Major pectoral flap harvest.*

*Oral Diseases*

**150**

**Figure 7.**

*(a) ALT flap design and (b) ALT flap harvest.*

**Figure 6.**

primary and salvage surgery (**Figure 8**). This flap based on the thoracoacromial artery can be raised as a myocutaneous or fasciocutaneous flap. It is reliable, robust, and easily harvested in terms to tongue reconstruction and can provide muscle and skin to fulfill the tongue and floor of the mouth and effectively separate the oral cavity from the neck. It must be suspended across the mandibular arch by either suturing

*(a) Tongue defect after resection, (b) RFFF harvest and (c) RFFF insetting.*

to the pterygoid musculature or securing to the mandible using drill holes to avoid and prevent the flap from falling [26]. This flap is considered a horse battle in rescue setting when a free flap fails. When the defect includes mandible, during the reconstruction it must have keep in mind that mandible contributes to airway stability, oral competence, speech, deglutition and mastication, so the goal of this reconstruction must include the preservation of the ability to open the mouth, occlusion, and the restoration of the inter arch continuity solutions to promote dental implants and restore chewing as mentioned in floor of mouth defects extended to mandibula. Not reconstructing the central defects will conclude in loss of the lip support with Andy Gump deformity, and not reconstructing the lateral defects will cause malocclusion and lateral shift in the position of mandible, so any intent must be done to reconstruct the mandible. Options in reconstruction include metal plates (**Figure 7**), non-vascularized bone grafts, osteomyocutaneous pedicled flaps, and osteocutaneous free flaps. Fixing soft tissues just with plates was widely used in the past and usually results in extrusion intraorally, external exposure or fracture of the plate up to 60% of the cases with a worst defect and a very poor functional outcome [27]. Autogenous bone grafts from iliac crest, scapula, or calvarium usually end in no vascularization of the new bone and its atrophy even more if radiation is added to the treatment, and finally similar results as the plating alone are obtained, so similarly they are no more used.

Currently the gold standard in mandible reconstruction is the osteocutaneous free flaps (**Figure 9a**–**c**) and carries the same consideration as mentioned in floor of mouth reconstruction with a trend to perform a first time micro vascularized bone reconstruction with dental implants mainly in a previous dentulous young patient [28]. In an aged edentulous patient in the reconstruction setting, there is most likely no need to be aware for dental implants unlike dentulous young patient. Again, in selected patients with poor clinical condition and not suitable for a long procedure, a osteocutaneous pediculate flap such as a osteomyocutaneous trapezius flap [29] or a bicortical parietal osteofascial pedicled flap [30] can be perform providing a better functional result compared with just soft tissue coverage. Both flaps require experience, skills, and anatomic knowledge to harvest them in a short period of time but are an excellent alternative when needed.

#### **4.3 Cheek**

The cheek resection is done less frequently except in some countries like India, where cheek cancer is frequent and as a consequence of chewing tobacco; usually its oncological resections leave a complex defect that includes skin and mucosa in an area where a functional lip is required to avoid food spillage. The consequent

defect may be small or big and simple or composite associated to another oral cavity subsite resection. Small lesions of the cheek could be let alone to epithelize, but a bigger one will end in a scar and retraction, so a reconstruction must be done. In most of the cases a facial artery mucomucosal flap (FAMM) could be used. This flap based on a branch of the facial artery is elevated in the layer underneath the facial artery including the overlying buccinators muscle and a small portion of orbicularis oris muscle close to the oral commissure; it is rotated to cover the defect commonly restoring it, and the donor site could be primary closed or let it to heal secondarily without impairing its final functional result. A huge defect might need a pediculate flap such as submandibular or supraclavicular flap or even a microvascular free flap. Some encourage for the supraclavicular pediculate flap as the first option in this scenery, which usually provides a good amount of a non-bulky tissue without affecting oncological resection of node neck dissection in level Ia and Ib, and adducing that submandibular flap is too bulky to placed it in this specific region.

#### **4.4 Hard palate**

The extent of resection of hard palate is crucial to define the type and modality of reconstruction. The defect may be small and involve any portion of the hard palate, the premaxilla, or any portion of the maxillary alveolus with or without tooth-bearing or may be as huge as more than 50% of the hard palate. Many of the times, it is associated with partial or total maxillectomy so ending in a complex defect. Small defects can be let just to re-epithelize with excellent results. For a bigger one, a skin graft can be used; the problem is to support it long enough to achieve its integration to the hard palate; sometimes, the flap is detached and lost in which case healing by second intention is required. Small to medium defects may demand to harvest a palatal mucoperiosteal flap (PMPF). This flap is based on the greater palatine artery; preserving this vascular pedicle allows to rotate

**153**

**Figure 10.**

*palate outcome after 2 years of reconstruction.*

*Modalities and State of Art in Oral Cancer Reconstruction*

it to resurface the mucosal defect [31]. Its limit is related to the amount of tissue needed, and up to 3 cm can be covered with this flap. In a bigger 3–5 cm hole, also a submandibular pediculate flap could be used to cover it. In as much as in this location, there are no specific needs for muscle or for a thicker soft tissue; any attempt should be done to assemble it with just enough muscle behind that guarantees skin perfusion by perforants preventing necrosis and providing a flat new tissue. A composite defect that includes the maxillary alveolus with tooth-bearing or partial to total maxillectomy will end in oroantral communication (**Figure 10a** and **b**). This type of reconstruction needs special considerations that are not the subject of this chapter and are best described in midface reconstruction; in general terms the main goal of the reconstruction is to restore chewing and solve the oroantral communication, so options for small include lesions and the use of an obturator that covers the opening avoiding leaks through the paranasal sinus and improving chew. As the aperture gets bigger, soft tissue flaps like a radial forearm free flap or an anterolateral thigh free flap are needed [32], and if dental implants are planned, microvascular osteocutaneous flaps obtained from fibula free flap or

*(a) Hard palate defect after resection, (b) hard palate outcome after 1 month reconstruction and (c) hard*

*DOI: http://dx.doi.org/10.5772/intechopen.91049*

iliac crest free flap must be designed.

#### *Modalities and State of Art in Oral Cancer Reconstruction DOI: http://dx.doi.org/10.5772/intechopen.91049*

*Oral Diseases*

defect may be small or big and simple or composite associated to another oral cavity subsite resection. Small lesions of the cheek could be let alone to epithelize, but a bigger one will end in a scar and retraction, so a reconstruction must be done. In most of the cases a facial artery mucomucosal flap (FAMM) could be used. This flap based on a branch of the facial artery is elevated in the layer underneath the facial artery including the overlying buccinators muscle and a small portion of orbicularis oris muscle close to the oral commissure; it is rotated to cover the defect commonly restoring it, and the donor site could be primary closed or let it to heal secondarily without impairing its final functional result. A huge defect might need a pediculate flap such as submandibular or supraclavicular flap or even a microvascular free flap. Some encourage for the supraclavicular pediculate flap as the first option in this scenery, which usually provides a good amount of a non-bulky tissue without affecting oncological resection of node neck dissection in level Ia and Ib, and adducing that submandibular flap is too bulky to placed it in this specific region.

*(a) FFF harvest, (b) FFF insetting and (c) FFF early postoperative outcome.*

The extent of resection of hard palate is crucial to define the type and modality of reconstruction. The defect may be small and involve any portion of the hard palate, the premaxilla, or any portion of the maxillary alveolus with or without tooth-bearing or may be as huge as more than 50% of the hard palate. Many of the times, it is associated with partial or total maxillectomy so ending in a complex defect. Small defects can be let just to re-epithelize with excellent results. For a bigger one, a skin graft can be used; the problem is to support it long enough to achieve its integration to the hard palate; sometimes, the flap is detached and lost in which case healing by second intention is required. Small to medium defects may demand to harvest a palatal mucoperiosteal flap (PMPF). This flap is based on the greater palatine artery; preserving this vascular pedicle allows to rotate

**152**

**4.4 Hard palate**

**Figure 9.**

it to resurface the mucosal defect [31]. Its limit is related to the amount of tissue needed, and up to 3 cm can be covered with this flap. In a bigger 3–5 cm hole, also a submandibular pediculate flap could be used to cover it. In as much as in this location, there are no specific needs for muscle or for a thicker soft tissue; any attempt should be done to assemble it with just enough muscle behind that guarantees skin perfusion by perforants preventing necrosis and providing a flat new tissue. A composite defect that includes the maxillary alveolus with tooth-bearing or partial to total maxillectomy will end in oroantral communication (**Figure 10a** and **b**). This type of reconstruction needs special considerations that are not the subject of this chapter and are best described in midface reconstruction; in general terms the main goal of the reconstruction is to restore chewing and solve the oroantral communication, so options for small include lesions and the use of an obturator that covers the opening avoiding leaks through the paranasal sinus and improving chew. As the aperture gets bigger, soft tissue flaps like a radial forearm free flap or an anterolateral thigh free flap are needed [32], and if dental implants are planned, microvascular osteocutaneous flaps obtained from fibula free flap or iliac crest free flap must be designed.

#### **Figure 10.**

*(a) Hard palate defect after resection, (b) hard palate outcome after 1 month reconstruction and (c) hard palate outcome after 2 years of reconstruction.*


**About the Editor**

## **Josip Lorincz**

Josip Lorincz received a B.Sc. (M.S. equivalent) and a Ph.D. degree in telecommunications engineering and computer science from the University of Split, Croatia, in 2002 and 2010, respectively. In 2003, he joined the Faculty of electrical engineering, mechanical engineering, and naval architecture, at the University of Split, Croatia, where he currently works as an associate professor. In the academic year 2009/2010, he was a visiting researcher at the Department of Electronics, informatics, and bioengineering of the Politecnico di Milano, Italy. As a project leader or researcher, he participated in more than thirty scientific and professional projects funded by the EU, public or private sector. He is the founder and chair of the Symposium on Green Networking and Computing, organized in the frame of the International Conference on Software, Telecommunications and Computer Networks (SoftCOM). He also serves as the technical program committee member for many international scientific conferences and as a reviewer for top scientific journals. He was a guest editor in different scientific journals and an evaluator of international scientific projects. His current research interests include energy-efficient wireless and wired networks, optimization in telecommunications, advanced design, management and analyses of sensor networks, and evolution of routing protocols. He has authored more than 60 research papers published in different scientific conferences and journals. He is a senior IEEE member, senior ACM member, associate member of the Croatian Academy of Engineering, and the first president of the Croatian ACM chapter. Since 2004, he has owned Cisco CCNA, CCAI, and BCMSN certificates. In 2012, he received an award for scientific work from his home institution, FESB, and in 2013, he was awarded as an outstanding young researcher by the Croatian Academy of Engineering. In 2023, from his home University of Split, he received a science award for present achievements in science.


*3.4.3 Forecasting block of ASS processes*

Corresponding software tools are described by mathematical model of park state changes amount of FMP and CP during fixed period of exploitation, materialtechnical technical service and types of repairs.

Output forecasting characteristics are the following:


**Abstract**

The Moyamoya disease [MMD] is a cereberovascular disorder characterized by progressive stenosis of intracranial internal carotid arteries and compensatory collateral formation at the base of the brain, mainly around the circle of Willis. When no particular associated risk factors can be identified, it is termed as Moyamoya disease. However, it may be associated with other neurological and extra- neurological disorders where it is termed as Moyamoya syndrome [MMS]. The condition is predominantly seen in East Asia and has bimodal age of distribution. The clinical manifestations are also age dependant with ischemia predominating in childhood and hemorrhagic manifestations being more common in adults. The pathogenesis is not entirely known, but genetic susceptibility is believed to be an important predisposing factor. The Suzuki staging system is most widely used for evaluation and staging of Moyamoya disease. The gold standard diagnostic modality is cerebral angiography but magnetic resonance imaging [MRA] has also been employed for diagnosis. Treatment is primarily surgical revascularization which is of 3 types: direct, indirect or combined revascularization. Although the role of revascularization surgery has been well established for ischemic MMD, the ideal surgical approach and the role of surgery in hemorrhagic MMD remains controversial.

**Keywords:** moyamoya disease, moyamoya syndrome, stroke, cerebral angiography, revascularization


K. Satué and J.C. Gardon

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63741

#### **Abstract**

In cyclic mares, the uterine environment can easily disturbed due to inflammatory proc‐ esses that occur secondary to microbial invasion. Different aerobic and anaerobic bacteria can enter the uterus during natural mating, artificial insemination, reproductive examina‐ tion or parturition. The postpartum period is a critical phase since due to relaxation of the uterus and cervix may favor recurrent infections air intake (pneumovagina) or urine col‐ lection in mares with poor perineal conformation. Infections are mainly caused by oppor‐ tunistic or commensal microorganisms, such as *Streptococcus zooepidemicus, hemolytic Escherichia coli* and *Staphylococcus aureus*. Other microorganisms like *Taylorella equigenita‐ lis, Klebsiella pneumoniae* and *Pseudomona aeruginosa* are transmitted through venereal route. Regarding to the fungal endometritis, the most common fungi include *Candida* and *Aspergillus*. These microorganisms cause infertility as a result of repeated inseminations during the breeding season and proliferate when the natural immune system is weak‐ ened in mares with advanced age and multiparous, or after repeated use of antibiotics. Indeed, in susceptible mare to endometritis, uterine defense mechanisms involving phag‐ ocytosis and opsonization by neutrophils, local synthesis of antibodies, mucociliary activ‐ ity, vascular and myoelectric activity permeability are compromised, leading to fluid accumulation in response to inflammation and infertility.

**Keywords:** cycling mare, infectious diseases, reproduction

#### **1. Introduction**

The ability to maintain a uterine environment compatible with embryonic and fetal life is essential for reproductive efficiency in horses. However, the uterine environment is easily disturbed by an inflammatory process following aerobic and anaerobic bacterial invasion, which can occur during natural breeding, artificial insemination (AI), reproductive examina‐ tion, parturition, postnatal infection (pneumovaginal infections), and mostly during a delayed postnatal cleaning of the uterus as a result of poor conformation [1].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

These bacterial uterine infections cause conception and embryo survival failures despite many repeated breeding during the season. In the horse industry, endometritis due to uterine infectious of bacterial origin brings about 25–60% economic losses, leading to infertility. In pregnant mares, these infectious diseases can appear as failure to conceive, early fetal losses, mid-gestational abortion, placentitis, birth of a septic neonate, postpartum metritis, or delays in rebreeding and can be related with other systemic pathologies of viral, bacterial, and fungal origin [2]. Besides in nonpregnant mares, these infections are most often caused by opportun‐ istic or commensal and venereal microorganisms, with a large variety of species have been isolated.

The healthy mare has three functional genital seals forming a barrier between the external environment and the uterine lumen [3, 4].

If the closure of the vulva is compromised, it results in the development of a pneumovagina with consequent aspiration of bacteria and other contaminated products [5]. The initial development of vaginitis can result in cervicitis and acute endometritis and therefore can lead subfertility. Usually, in mares, contamination of the caudal reproductive tract with bacteria during pregnancy can result in embryonic death. In late pregnancy, it can result in the development of placentitis and abortion [4, 6].

The more severe conformational abnormalities result in failure of the vulval closing, increasing fecal contamination since the vulva forms a platform in which feces can collect. In these cases, the vulval lips are angled at 25° for even 50° to the vertical. According to Bradecamp [6], defective vulval conformation can be:

**•** Congenital, rare to find.

These bacterial uterine infections cause conception and embryo survival failures despite many repeated breeding during the season. In the horse industry, endometritis due to uterine infectious of bacterial origin brings about 25–60% economic losses, leading to infertility. In pregnant mares, these infectious diseases can appear as failure to conceive, early fetal losses, mid-gestational abortion, placentitis, birth of a septic neonate, postpartum metritis, or delays in rebreeding and can be related with other systemic pathologies of viral, bacterial, and fungal origin [2]. Besides in nonpregnant mares, these infections are most often caused by opportun‐ istic or commensal and venereal microorganisms, with a large variety of species have been

The healthy mare has three functional genital seals forming a barrier between the external

**•** Lips of vulva: examination of vulvar and perineal conformation should be included in all prebreeding examinations or evaluations for infertility. The vulva should be a vertical position aligned with the anal opening. The labia should be tight, with most of length below the tuber ischii. The vulvar lips may become parted as a consequence of malformation caused by previous traumatic injuries. In older multiparous mares, there is a tendency for extreme relaxation of the vulvar lips, particularly during estrus, as well as tilting of the dorsal aspect of the vulva becomes horizontal as it is pulled cranially over the tuber ischii. These anatomic changes predispose the mare to pneumavagina (windsucking) and pneu‐ mouterus, ultimately causing urine to pool in the cranial vagina producing ascending infection and contaminate the uterus when the cervix is open. Pneumovagina may lead to an urovagina (urine pooling within the vagina) when the vestibule and urethral opening are displaced cranially. In this situation, contamination with fecal material adds to the

**•** Vulvovaginal constriction: immediately in front of the external urethral opening is the vulvovaginal constriction or vestibular seal. In genitally healthy mares, this forms the second line of defense against aspirated air and fecal material. In a normal mare, the vestibulovaginal area remains sealed even when the vulvar labia are parted. Compromised vestibulovaginal sphincter function is suspected when air is sucked into the vagina of

**•** Cervix: forms the important third (and last) protective physical barrier to protect the uterus from the external environment. The cervix must also relax during estrus to allow intrauter‐ ine ejaculation or insemination of semen and drainage of uterine fluid, late pregnancy, and the immediate postpartum period. An inflammation of the cervix is usually associated with

If the closure of the vulva is compromised, it results in the development of a pneumovagina with consequent aspiration of bacteria and other contaminated products [5]. The initial development of vaginitis can result in cervicitis and acute endometritis and therefore can lead subfertility. Usually, in mares, contamination of the caudal reproductive tract with bacteria during pregnancy can result in embryonic death. In late pregnancy, it can result in the

compromised secondary to rectovaginal tears and other foaling injuries.

isolated.

258 Genital Infections and Infertility

environment and the uterine lumen [3, 4].

increased risk of infection.

endometritis and/or vaginitis.

development of placentitis and abortion [4, 6].

**•** Acquired, which is due to vulval enlarging resulting for the repeated foaling, injury to perineal tissue and poor body condition (old, thin mares). Older and pluriparous mares are frequently affected with pneumovagina. However, in cases of young mares that are in work and have little body fat and/or poor vulval conformation, it can develop these pathology.

Pneumovagina might occur during estrus when the perineal tissues are more relaxed. Some mares make a noise while walking; however, the diagnosis of other mares may be difficult by the absence of noises. The pathognomonic sign is the presence of hyperemia and a frothy exudate in the anterior vagina, on examination with a speculum. Poor cervical dilation may reduce uterine clearance, while cervical damage or incompetence may predispose to infection. Finally, susceptible mares may accumulate more fluid within their uterus than normal mares because of a greater production of glandular secretions secondary to inflammation. The consequence of decreased physical clearance is that bacteria, inflammatory by-products and fluid remain in the uterine lumen, inducing more inflammation, mucosal cell damage, and fibrosis. The persistent inflammation may result in premature luteolysis. Ultimately, chronic inflammation results in degenerative changes within the endometrium and reduced fertility [5–7].

Real-time ultrasound examination of the uterus may reveal the presence of air or urine as hyperechoic or hypoechoic foci seen at the opposed luminal surfaces, respectively. Cytological and histological examination of the endometrium may demonstrate significant numbers of neutrophils indicative of an endometritis [6]. The clinical presentation of acute or chronic reproductive tract infections in the mare is salpingitis, cervicitis, and vaginitis [2, 5].


*Article* **A New Method to Decision-Making with Fuzzy Competition Hypergraphs**

#### **Musavarah Sarwar 1, Muhammad Akram 2,\* and Noura Omair Alshehri 3**

Received: 23 July 2018; Accepted: 12 September 2018; Published: 16 September 2018

**Abstract:** Hypergraph theory is the most developed tool for demonstrating various practical problems in different domains of science and technology. Sometimes, information in a network model is uncertain and vague in nature. In this paper, our main focus is to apply the powerful methodology of fuzziness to generalize the notion of competition hypergraphs and fuzzy competition graphs. We introduce various new concepts, including fuzzy column hypergraphs, fuzzy row hypergraphs, fuzzy competition hypergraphs, fuzzy *k*-competition hypergraphs and fuzzy neighbourhood hypergraphs, strong hyperedges, *k*th strength of competition and symmetric properties. We design certain algorithms for constructing different types of fuzzy competition hypergraphs. We also present applications of fuzzy competition hypergraphs in decision support systems, including predator–prey relations in ecological niche, social networks and business marketing.

**Keywords:** fuzzy competition hypergraph; fuzzy *k*-competition hypergraph; fuzzy open neighbourhood; fuzzy closed neighbourhood; ecological niches

**MSC:** 05C65; 05C85; 68R10; 03E72