Neocortical developmental analysis of vasculature and their growth factors offer new insight into fragile X syndrome abnormalities

Fragile X Syndrome (FXS) is the most common single gene cause for Autism Spectrum Disorder and the most prevalent form of inherited mental retardation. Our prior studies have demonstrated that adult FXS mice have abnormal blood vessel density (BVD) and elevated Vascular Endothelial Growth Factor A expression (VEGF‐A). VEGF‐A is one of the most prominent regulators of BVD, and its abnormal expression is the most likely cause for FXS BVD abnormalities. We have demonstrated that attenuating elevated VEGF‐A expression can ameliorate many non‐vascular FXS abnormalities (Belagodu, Zendeli Slater and Galvez: Dev Neurobiol 77 (2017) 14–25), suggesting that abnormal VEGF‐A expression is an underlying cause for some FXS abnormalities. However, FXS is a developmental disorder and VEGF‐A's potential role in mediating FXS abnormalities during development have never been explored. Furthermore, VEGF‐A is one protein in a family of proteins (VEGF‐A, VEGF‐B, VEGF‐C, VEGF‐D, & PLGF) that activate one of three primary receptors (VEGFR1, VEGFR2, & VEGFR3). Abnormal expression of any of these proteins could hinder proper development. The current study demonstrated that FXS mice do not exhibit normal BVD developmental patterns, resulting in elevated adult expression, most likely due to observed elevated VEGF‐A adult expression. Interestingly, all five VEGF family of proteins exhibited altered developmental expression patterns that could cause abnormal development. However, none of the receptors exhibited abnormal adult expression, but did exhibit altered developmental expression. Expanding upon our prior analyses, the current study provides additional interesting insight towards potential developmental mechanisms mediating FXS abnormalities, while offering further sites for age specific therapeutic interventions. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1321–1333, 2017     

Exposure to high‐concentration oxygen in the neonatal period induces abnormal retinal vascular patterning in mice

The interruption of vascular development could cause structural and functional abnormalities in tissues. We have previously reported that short‐term treatment of newborn mice with vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitors induces abnormal retinal vascular growth and patterns. An exposure of neonatal mice to high‐concentration oxygen disturbs normal retinal vascular development. The present study aimed to determine (1) whether vascular abnormalities are observed in the retina of newborn mice exposed to high concentrations of oxygen, and (2) how astrocyte network formation is affected following the exposure to hyperoxia. Newborn (postnatal day 0) mice were exposed to 75% oxygen for 48 or 96 hr. During hyperoxia exposure, VEGF expression decreased, and the onset of retinal vascularization was completely suppressed. After completion of the hyperoxic period, retinal vascularization occurred, but it was delayed in a hyperoxic exposure duration‐dependent manner. In retinas of hyperoxia‐exposed mice, dense capillary plexuses were found, and the number of arteries and veins decreased. The astrocyte network formation was slightly delayed under hyperoxic conditions, and the network became denser in retinas of mice with an episode of hyperoxia. Expression of VEGF levels in the avascular retina of mice that were exposed to hyperoxia was higher than that of control mice. These results suggest that short‐term interruption of the onset of vascular development resulting from the reduction in VEGF signals induces abnormal vascular patterns in the mouse retina. The abnormalities in retinal astrocyte behavior might contribute to the formation of an abnormal retinal vascular growth.     

Bevacizumab and ranibizumab on microvascular endothelial cells: A comparative study

Given its broad effects in endothelium, vascular endothelial growth factor (VEGF) represents the primary rate‐limiting step of angiogenesis. Therefore, VEGF targeting therapies were soon developed. Bevacizumab and ranibizumab are two of these therapeutic agents already in clinical use. Bevacizumab was first used for cancer treatment, whereas ranibizumab was designed to target choroidal neovascularization, the main cause of blindness in age‐related macular degeneration. The present study aims to compare the multiple effects of bevacizumab and ranibizumab in human microvascular endothelial cells (HMECs). HMEC cultures were established and treated during 24 h with the anti‐VEGF agents within the intravitreal‐established concentration range or excipients. Analyses of VEGF content in cell media and VEGF receptor‐2 (VEGFR‐2) expression in cell lysates were performed. No cell cytotoxicity (MTS assay) was found in anti‐VEGF‐treated cultures at any concentration. Apoptosis (TUNEL assay) was significantly increased and cell proliferation (BrdU assay), migration (transwell assay) and assembly into vascular structures were significantly reduced by incubation with both agents at the two doses used. These findings were accompanied by a strong decrease in VEGF release, and in phosphorylated VEGFR‐2 and Akt expression for both agents at the clinical concentration. Interestingly, phosphorylated Erk was only significantly reduced upon bevacizumab treatment. In addition, proliferation was more affected by ranibizumab, whereas migration, capillary formation, and phosphorylated VEGFR2 expression were significantly reduced by bevacizumab as compared to ranibizumab. Therefore, although both agents presented anti‐angiogenic actions, distinct effects were exerted by the two molecules in HMEC. These findings suggest that a careful confirmation of these effects in clinical settings is mandatory. J. Cell. Biochem. 108: 1410–1417, 2009. © 2009 Wiley‐Liss, Inc.     

FAK contributes to proteinuria in hypercholesterolaemic rats and modulates podocyte F‐actin re‐organization via activating p38 in response to ox‐LDL

Focal adhesion kinase (FAK) is a non‐receptor protein tyrosine kinase that regulates cell adhesion, proliferation and differentiation. In the present study, a rat model of high fat diet‐induced hypercholesterolaemia was established to investigate the involvement of FAK in lipid disorder‐related kidney diseases. We showed focal fusion of podocyte foot process that occurred at as early as 4 weeks in rats consuming high fat diet, preceding the onset of proteinuria when aberrant phosphorylation of FAK was found. These abnormalities were ameliorated by dietary intervention of TAE226, a reported inhibitor of FAK. FAK is also an adaptor protein initiating cascades of intracellular signals including c‐Src, Rho GTPase and mitogen‐activated protein kinase (MAPK). P38 MAPK belongs to the latter and is centrally involved in kidney diseases. Our cell culture data revealed oxidized low‐density lipoprotein (ox‐LDL) triggered hyper‐phosphorylation of FAK and p38, ectopic expression of cellular markers (manifested as decreased WT1, podocin and NEPH1, and increased vimentin and mmp9), and re‐arrangement of F‐actin filaments with enhanced cell motility; these mutations were significantly rectified by FAK shRNA. Notably, pre‐treatment of p38 inhibitor did not alter FAK activation, albeit its deletion of p38 hyper‐activity and attenuation of cellular abnormalities, demonstrating that p38 acted as a downstream effector of FAK signalling and ox‐LDL damaged podocytes in a FAK/p38‐dependent manner. This was further identified by animal data that p38 activation was also abrogated by TAE226 treatment in hypercholesterolaemic rats, suggesting that FAK/p38 axis might also be involved in in vivo events. These findings provided a potential early mechanism of hypercholesterolaemia‐related podocyte damage and proteinuria.     

Changes in VEGF expression and DNA synthesis in hepatocytes from hepatectomized and tumour‐bearing mice

Transplanted tumours could modify the intensity and temporal distribution of the cellular proliferation in normal cell populations, and partial hepatectomy alters the serum concentrations of substances involved in cellular proliferation, leading to the compensatory liver hyperplasia. The following experiments were designed in order to study the SI (S‐phase index) and VEGF (vascular endothelial growth factor) expression in regenerating liver (after partial hepatectomy) of adult male mice bearing a hepatocellular carcinoma, throughout one complete circadian cycle. We used adult male C3H/S‐strain mice. After an appropriate period of synchronization, the C3H/S‐histocompatible ES2a hepatocellular carcinoma was grafted into the subcutaneous tissue of each animal's flank. To determine the index of SI and VEGF expression of hepatocytes, we used immunohistochemistry. The animals were divided into two experimental groups: Group I, control, hepatectomized animals; Group II, hepatectomized tumour‐bearing animals. The statistical analysis of SI and VEGF expression was performed using Anova and Tukey as a postcomparison test. The results show that in the second group, the curve of SI changes the time points for maximum and minimum activity, and the peak of VEGF expression appears before the first group. In conclusion, in the hepatectomized mice, the increases of hepatic proliferation, measured by the SI index, may produce a rise in VEGF expression with the object of generating a vascular network for hepatic regeneration. Lastly, as we have mentioned, in hepatectomized and tumour‐bearing mice, the peak of VEGF expression appears before the one of DNA synthesis.     

Oestrogen‐induced angiogenesis promotes adenomyosis by activating the Slug‐VEGF axis in endometrial epithelial cells

Adenomyosis is an oestrogen‐dependent disease characterized by the invasion of endometrial epithelial cells into the myometrium of uterus, and angiogenesis is thought to be required for the implantation of endometrial glandular tissues during the adenomyotic pathogenesis. In this study, we demonstrate that compared with eutopic endometria, adenomyotic lesions exhibited increased vascularity as detected by sonography. Microscopically, the lesions also exhibited an oestrogen‐associated elevation of microvascular density and VEGF expression in endometrial epithelial cells. We previously reported that oestrogen‐induced Slug expression was critical for endometrial epithelial–mesenchymal transition and development of adenomyosis. Our present studies demonstrated that estradiol (E2) elicited a Slug‐VEGF axis in endometrial epithelial cells, and also induced pro‐angiogenic activity in vascular endothelial cells. The antagonizing agents against E2 or VEGF suppressed endothelial cells migration and tubal formation. Animal experiments furthermore confirmed that blockage of E2 or VEGF was efficient to attenuate the implantation of adenomyotic lesions. These results highlight the importance of oestrogen‐induced angiogenesis in adenomyosis development and provide a potential strategy for treating adenomyosis through intercepting the E2‐Slug‐VEGF pathway.     

Bevacizumab Attenuates Hepatic Fibrosis in Rats by Inhibiting Activation of Hepatic Stellate Cells

Angiogenesis is a fundamental part of the response to tissue injury, which is involved in the development of hepatic fibrosis. Vascular endothelial growth factor plays an important role in angiogenesis. The expression of VEGF is increased during hepatic fibrogenesis and correlates with the micro-vessel density. In this study, we investigated the effects of bevacizumab, an anti-angiogenetic drug, on the formation of hepatic fibrosis. We found that bevacizumab could attenuate the development of hepatic fibrosis and contribute to the protection of liver function. Bevacizumab was also found to downregulate the expression α-SMA and TGF-β1, which have been reported to be profibrogenic genes in vivo. We also observed that the expression of VEGF increased significantly during the development of hepatic fibrosis and CCl₄ was found to induce hepatocytes to secrete VEGF, which led to the activation and proliferation of HSCs. Bevacizumab was also found to block the effects of the hepatocytes on the activation and proliferation of HSCs. Our results suggest that bevacizumab might alleviate liver fibrosis by blocking the effect of VEGF on HSCs. Bevacizumab might be suitable as a potential agent for hepatic fibrosis therapy.     

In vitro effects of 2‐methoxyestradiol‐bis‐sulphamate on the non‐tumorigenic MCF‐12A cell line

A priority in recent anti‐cancer drug development has been attaining better side‐effect profiles for potential compounds. To produce highly specific cancer therapies it is necessary to understand both the effects of the proposed compound on cancer and on normal cells comprising the rest of the human body. Thus in vitro evaluation of these compounds against non‐carcinogenic cell lines is of critical importance. One of the most recent developments in experimental anti‐cancer agents is 2‐methoxyestradiol‐bis‐sulphamate (2ME‐BM), a sulphamoylated derivative of 2‐methoxyestradiol. The aim of this study was to evaluate the in vitro effects of 2ME‐BM on cell proliferation, morphology and mechanisms of cell death in the non‐carcinogenic MCF‐12A breast epithelial cell line. The study revealed changes in proliferative capacity, morphology and cell death induction in response to 2ME‐BM exposure (24 h at 0.4 µM). Microscopy showed decreased cell density and cell death‐associated morphology (increased apoptotic characteristics), a slight increase in acidic intracellular vesicles and insignificant ultra‐structural aberrations. Mitotic indices revealed a G₂M‐phase cell cycle block. This was confirmed by flow cytometry, where an increased fraction of abnormal cells and a decrease in cyclin B1 levels were observed. These results evidently demonstrate that the non‐carcinogenic MCF‐12A cell line is less susceptible when compared to 2ME‐BM‐exposed cancer cell lines previously tested. Further in vitro research into the mechanism of this potentially useful compound is warranted. Copyright © 2010 John Wiley & Sons, Ltd.     

Enhanced expression of the Flt‐1 and Flk‐1 receptor tyrosine kinases in a newborn piglet model of ischemic tolerance

Vascular endothelial growth factor (VEGF), an angiogenic factor induced by hypoxia, also exerts direct effects on neural tissues. VEGF up‐regulation after hypoxia coincides with expression of its two tyrosine kinase receptors Flt‐1(VEGFR‐1) and Flk‐1 (KDR/VEGFR‐2), which are the key mediators of physiological angiogenesis. We have recently shown that hypoxic‐preconditioning (PC) leading to tolerance to hypoxia–ischemia in neonatal piglet brain resulted in increased expression of VEGF. In this study, we used a hypoxic‐preconditioning model of ischemic tolerance to analyze the expression and cellular distribution of VEGF receptors and phosphorylation of cAMP‐response element‐binding protein (CREB) in newborn piglet brain. The response of Flt‐1 and Flk‐1 mRNA to PC alone was biphasic with peaks early (6 h) and late (1 week) after PC. The mRNA expression of Flt‐1 and Flk‐1 in piglets preconditioned 24 h prior to hypoxia–ischemia was significantly higher than non‐preconditioned piglets and remained up‐regulated up to 7 days. Furthermore, PC prior to hypoxia–ischemia significantly increased the protein levels of Flt‐1 and Flk‐1 compared with hypoxia–ischemia in a time‐dependent manner. Double‐immunolabeling indicated that both Flt‐1 and Flk‐1 are expressed in neurons and endothelial cells with a similar time course of expression following PC and that PC leads to the growth of new vessels. Finally, our data demonstrate that PC significantly phosphorylated and activated cAMP‐response element‐binding protein in nucleus. These results suggest that mechanism(s) initiated by PC can induce VEGF receptor up‐regulation in newborn brain and that VEGF–VEGF receptor‐coupled signal transduction pathways could contribute to the establishment of tolerance following hypoxia–ischemia.     

Gax suppresses chemerin/CMKLR1‐induced preadipocyte biofunctions through the inhibition of Akt/mTOR and ERK signaling pathways

Adipose tissue is closely associated with angiogenesis and vascular remodeling. Chemerin is involved in inflammatory reaction and vascular dysfunction. However, the mechanisms of chemerin participating in vascular remodeling and whether Growth arrest‐specific homeobox (Gax) can effectively intervene it remain obscured. Here, 3T3‐F442A preadipocytes were cultured, injected into athymic mice to model fat pads, and treated respectively with Ad‐chemerin, Ad‐Gax, or specific inhibitors in vitro and in vivo. MTT, flow cytometry, Western blotting, and imunohisto(cyto)‐chemistry analyses showed that chemerin enhanced the expression of FABP4 and VEGF, activated Akt/mTOR and ERK pathways, increased the cell percent of S phase, decreased the percent of G0‐G1 phase and apoptotic cells, and augmented neovascular density in fat pads. Inversely, Gax suppressed the expression of these adipogenic and vasifactive markers and these signaling proteins, decreased the percent of S phase cells, and increased those of G0‐G1 phase and apoptotic cells, and reduced the neovascular density. Our results indicate that chemerin‐CMKLR1 activates Akt/mTOR and ERK pathways and facilitates preadipocyte proliferation, adipogenesis, and angiogenesis. Contrarily, Gax weakens the effect of chemerin on preadipocyte biofunctions.     

Vascular endothelial growth factor prevents G93A‐SOD1‐induced motor neuron degeneration

Amyotrophic lateral sclerosis (ALS) is an adult‐onset neurodegenerative disorder characterized by selective loss of motor neurons (MNs). Twenty percent of familial ALS cases are associated with mutations in Cu²⁺/Zn²⁺ superoxide dismutase (SOD1). To specifically understand the cellular mechanisms underlying mutant SOD1 toxicity, we have established an in vitro model of ALS using rat primary MN cultures transfected with an adenoviral vector encoding a mutant SOD1, G93A‐SOD1. Transfected cells undergo axonal degeneration and alterations in biochemical responses characteristic of cell death such as activation of caspase‐3. Vascular endothelial growth factor (VEGF) is an angiogenic and neuroprotective growth factor that can increase axonal outgrowth, block neuronal apoptosis, and promote neurogenesis. Decreased VEGF gene expression in mice results in a phenotype similar to that seen in patients with ALS, thus linking loss of VEGF to the pathogenesis of MN degeneration. Decreased neurotrophic signals prior to and during disease progression may increase MN susceptibility to mutant SOD1‐induced toxicity. In this study, we demonstrate a decrease in VEGF and VEGFR2 levels in the spinal cord of G93A‐SOD1 ALS mice. Furthermore, in isolated MN cultures, VEGF alleviates the effects of G93A‐SOD1 toxicity and neuroprotection involves phosphatidylinositol 3‐kinase/protein kinase B (PI3K/Akt) signaling. Overall, these studies validate the usefulness of VEGF as a potential therapeutic factor for the treatment of ALS and give valuable insight into the responsible signaling pathways and mechanisms involved. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Effects of siRNA Targeting c‐Myc and VEGF on Human Colorectal Cancer Volo Cells

c‐Myc and vascular endothelial growth factor (VEGF) genes are frequently deregulated and overexpressed in this malignancy, and strategies designed to inhibit c‐Myc and VEGF expression in cancer cells may have considerable therapeutic value. In the present study, we design and use short interfering RNA (siRNA) to inhibit c‐Myc and VEGF expression in colorectal cancer Volo cells and validate their effects on cell proliferation, cell cycle, apoptosis, and cell metastasis. Upon transient transfection with plasmid‐encoding siRNA, it was found that expression of c‐Myc and VEGF was significantly downregulated in siRNA‐transfected cells and the downregulation of c‐Myc and VEGF inhibited cell growth and induced apoptosis and metastasis of Volo cells. c‐Myc and VEGF downregulation also increased cell population in the G0–G1 phase. In conclusion, the specific siRNA efficiently silenced the expression of c‐Myc and VEGF, further suppressed the cell proliferation, triggered cell apoptosis, and inhibited cell invasiveness of colorectal cancer Volo cells. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:499‐505, 2012;Viewthis article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21455     

CD151 mediates netrin‐1‐induced angiogenesis through the Src‐FAK‐Paxillin pathway

Crosstalk between the nervous and vascular systems is important during development and in response to injury, and the laminin‐like axonal guidance protein netrin‐1 has been studied for its involvement in angiogenesis and vascular remodelling. In this study, we examined the role of netrin‐1 in angiogenesis and explored the underlying mechanisms. The effect of netrin‐1 on brain tissues and endothelial cells was examined by immunohistochemistry and western blotting in a middle cerebral artery occlusion model and in human umbilical vein endothelial cells. Cell proliferation and cell cycle progression were assessed by the MTT assay and flow cytometry, and the Transwell and tube formation assays were used to examine endothelial cell motility and function. Netrin‐1 up‐regulated CD151 and VEGF concomitant with the activation of focal adhesion kinase (FAK), Src and Paxillin in vitro and in vivo and the induction of cell proliferation, migration and tube formation in vitro. Silencing of CD151 abolished the effects of netrin‐1 on promoting cell migration and tube formation mediated by the activation of FAK/Src signalling. Netrin‐1 promoted angiogenesis in vitro and in vivo by activating the FAK/Src/Paxillin signalling pathway through a mechanism dependent on the expression of the CD151 tetraspanin, suggesting the existence of a netrin‐1/FAK/Src/CD151 signalling axis involved in the modulation of angiogenesis.     

Fibroblast growth factor‐2/platelet‐derived growth factor enhances atherosclerotic plaque stability

Increased immature neovessels contribute to plaque growth and instability. Here, we investigated a method to establish functional and stable neovessel networks to increase plaque stability. Rabbits underwent aortic balloon injury and were divided into six groups: sham, vector and lentiviral transfection with vascular endothelial growth factor‐A (VEGF)‐A, fibroblast growth factor (FGF)‐2, platelet‐derived growth factor (PDGF)‐BB and FGF‐2 + PDGF‐BB. Lentivirus was percutaneously injected into the media‐adventitia of the abdominal aorta by intravascular ultrasound guidance, and plaque‐rupture rate, plaque‐vulnerability index and plaque neovessel density at the injection site were evaluated. Confocal microscopy, Prussian Blue assay, Evans Blue, immunofluorescence and transmission electron microscopy were used to assess neovessel function and pericyte coverage. To evaluate the effect of FGF‐2/PDGF‐BB on pericyte migration, we used the mesenchymal progenitor cell line 10T1/2 as an in vitro model. VEGF‐A‐ and FGF‐2‐overexpression increased the number of immature neovessels, which caused intraplaque haemorrhage and inflammatory cell infiltration, eventually resulting in the plaque vulnerability; however, FGF‐2/PDGF‐BB induced mature and functional neovessels, through increased neovessel pericyte coverage. Additionally, in vitro analysis of 10T1/2 cells revealed that FGF‐2/PDGF‐BB induced epsin‐2 expression and enhanced the VEGF receptor‐2 degradation, which negatively regulated pericyte function consistent with the in vivo data. These results showed that the combination of FGF‐2 and PDGF‐BB promoted the function and maturation of plaque neovessels, thereby representing a novel potential treatment strategy for vulnerable plaques.     

LncRNA TNK2‐AS1 regulated ox‐LDL‐stimulated HASMC proliferation and migration via modulating VEGFA and FGF1 expression by sponging miR‐150‐5p

Long non‐coding RNAs (lncRNAs) have been indicated for the regulatory roles in cardiovascular diseases. This study determined the expression of lncRNA TNK2 antisense RNA 1 (TNK2‐AS1) in oxidized low‐density lipoprotein (ox‐LDL)‐stimulated human aortic smooth muscle cells (HASMCs) and examined the mechanistic role of TNK2‐AS1 in the proliferation and migration of HASMCs. Our results demonstrated that ox‐LDL promoted HASMC proliferation and migration, and the enhanced proliferation and migration in ox‐LDL‐treated HASMCs were accompanied by the up‐regulation of TNK2‐AS1. In vitro functional studies showed that TNK2‐AS1 knockdown suppressed cell proliferation and migration of ox‐LDL‐stimulated HASMCs, while TNK2‐AS1 overexpression enhanced HASMC proliferation and migration. Additionally, TNK2‐AS1 inversely regulated miR‐150‐5p expression via acting as a competing endogenous RNA (ceRNA), and the enhanced effects of TNK2‐AS1 overexpression on HASMC proliferation and migration were attenuated by miR‐150‐5p overexpression. Moreover, miR‐150‐5p could target the 3’ untranslated regions of vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 1 (FGF1) to regulate FGF1 and VEGFA expression in HASMCs, and the inhibitory effects of miR‐150‐5p overexpression in ox‐LDL‐stimulated HASMCs were attenuated by enforced expression of VEGFA and FGF1. Enforced expression of VEGFA and FGF1 also partially restored the suppressed cell proliferation and migration induced by TNK2‐AS1 knockdown in ox‐LDL‐stimulated HASMCs, while the enhanced effects of TNK2‐AS1 overexpression on HASMC proliferation and migration were attenuated by the knockdown of VEGFA and FGF1. Collectively, our findings showed that TNK2‐AS1 exerted its action in ox‐LDL‐stimulated HASMCs via regulating VEGFA and FGF1 expression by acting as a ceRNA for miR‐150‐5p.     

Functional roles of angiogenic factors and receptors on non‐endothelial cells in the oropharyngeal cavity of the chukar partridge (Alectoris chukar)

The vascular endothelial growth factor (VEGF) family belong to the platelet‐derived growth factor supergene family and is involved in angiogenesis and mitogenesis. The VEGF–VEGFR system regulates endothelial cell proliferation, migration, vascular permeability, secretion and other non‐endothelial cells functions. To clarify the possible role of endothelial and non‐endothelial cells, VEGF and its receptors, vascular endothelial cell growth inhibitor (VEGI) were immunohistochemically examined in oropharyngeal organs. Ten adult partridges were used in this study and the pharynx and larynx were dissected together with the palate and tongue. VEGI, VEGF and its receptor were highly expressed in luminal epithelial and stromal cells, when compared to glandular epithelial and muscle cells (P < 0.05). Moreover, VEGF, its receptors and VEGI were expressed rather strongly in the endothelial cells of the blood capillaries and in both the endothelial and smooth muscle cells of the large and small blood vessels. In conclusion, VEGF and its receptors (flt1/fms, flk1/KDR and flt4) and VEGI were expressed by various cell groups at varying intensity in the oropharyngeal organs. This demonstrates that they play a critical role in the regulation and maintenance of the functions in cells different from endothelial ones as well as in cell proliferation, differentiation, apoptosis and angiogenesis.