Vascular Endothelial Growth Factor Supplementation Research Articles

A triple growth factor strategy for optimizing bone augmentation in mice.

With dental implant treatment becoming the gold standard, the need for effective bone augmentation prior to implantation has grown. This study aims to evaluate a bone augmentation strategy integrating three key growth factors: bone morphogenetic protein-2 (BMP-2), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor (VEGF). Collagen scaffolds incorporating BMP-2, IGF-1, or VEGF were fabricated and categorized into five groups based on their content: scaffold alone; BMP-2 alone (BMP-2); BMP-2 and IGF-1 (BI); BMP-2, IGF-1, and VEGF (BIV); and BMP-2 and IGF-1 with an earlier release of VEGF (BI + V). The prepared scaffolds were surgically implanted into the calvarias of C57BL/6JJcl mice, and hard tissue formation was assessed after 10 and 28 days through histological, tomographic, and biochemical analyses. The combination of BMP-2 and IGF-1 induced a greater volume of hard tissue augmentation compared with that of BMP-2 alone, regardless of VEGF supplementation, and these groups had increased levels of cartilage compared with others. The volume of hard tissue formation was greatest in the BIV group. In contrast, the BI + V group exhibited a hard tissue volume similar to that of the BI group. While VEGF and CD31 levels were highest in the BIV group at 10 days, there was no correlation at the same time point between hard tissue formation and the quantity of M2 macrophages. In conclusion, the simultaneous release of BMP-2, IGF-1, and VEGF proved to be effective in promoting bone augmentation.

Degrees of macrophage-facilitated healing in aneurysm occlusion devices.

Insufficient healing of aneurysms following treatment with vascular occlusion devices put patients at severe risk of fatal rupture. Therefore, promoting healing and not just occlusion is vital to enhance aneurysm healing. Following occlusion device implantation, healing is primarily orchestrated by macrophage immune cells, ending with fibroblasts depositing collagen to stabilize the aneurysm neck and dome, preventing rupture. Several modified occlusion devices are available currently on-market. Previous in vivo work demonstrated that modifications of occlusion devices with a shape memory polymer foam had enhanced aneurysm healing outcomes. To better understand cellular response to occlusion devices and improve aneurysm occlusion device design variables, we developed an in vitro assay to isolate prominent interactions between devices and key healing players: macrophages and fibroblasts. We used THP-1 monocyte derived macrophages and human dermal fibroblasts in our cell culture models. Macrophages were allowed device contact with on-market competitor aneurysm occlusion devices for up to 96 h, to allow for any spontaneous device-driven macrophage activation. Macrophage secreted factors were captured in the culture media, in response to device-specific activation. Fibroblasts were then exposed to device-conditioned macrophage media (with secreted factors alone), to determine if there were any device-induced changes in collagen secretion. Our in vitro studies were designed to test the direct effect of devices on macrophage activation, and the indirect effect of devices on collagen secretion by fibroblasts to promote aneurysm healing and stabilization. Over 96 h, macrophages displayed significant migration toward and interaction with all tested devices. As compared to other devices, shape memory polymer foams (SMM, Shape Memory Medical) induced significant changes in gene expression indicating a shift toward an anti-inflammatory pro-healing M2-like phenotype. Similarly, macrophages in contact with SMM devices secreted more vascular endothelial growth factor (VEGF) compared with other devices. Macrophage conditioned media from SMM-contacted macrophages actively promoted fibroblast secretion of collagen, comparable to amounts observed with exogenous stimulation via VEGF supplementation. Our data indicate that SMM devices may promote good aneurysm healing outcomes, because collagen production is an essential step to ultimately stabilize an aneurysm.

Orthotopic Ferret Tracheal Transplantation Using a Recellularized Bioengineered Graft Produces Functional Epithelia.

Tracheal grafts may be necessary to bridge long-segment defects after curative resection for airway obstructions. Bioengineered grafts have emerged as an appealing option, given the possibilities of altering the histologic and cellular profile of the conduit. We previously designed a bioreactor capable of luminally decellularizing and recellularizing a ferret trachea with surface airway epithelia (SAE) basal cells (BCs), and we sought to assess the fate of these grafts when transplanted in an orthotopic fashion. As adjuncts to the procedure, we investigated the use of a vascular endothelial growth factor (VEGF)-laden hydrogel and of immunosuppression (IS) in graft revascularization and viability. IS was shown to limit early graft revascularization, but this effect could be counteracted with VEGF supplementation. Submucosal gland (SMG) loss was shown to be inevitable regardless of the revascularization strategy. Lastly, the bioengineered tracheas survived one month after transplant with differentiation of our implanted BCs that then transitioned into a recipient-derived functional epithelium. The work presented in this manuscript has important implications for future cellular and regenerative therapies.

99. Aging Associated Loss of Vegfa Impairs Muscle Regeneration

Purpose: Global populations, particularly in developed countries, have continued to age. Notably, aging is intimately correlated with frailty, a parameter that has been previously demonstrated to correlate significantly with mortality and concomitantly correlate with loss of muscle mass. The molecular mechanisms behind aging associated impaired skeletal muscle regeneration remain incompletely understood. Our group previously demonstrated aging-associated loss of hypoxia signaling impairs skeletal muscle myogenic potential. Given these findings, we hypothesized vascular endothelial growth factor (VEGFA), a potent downstream signal well characterized in nascent vascular ingrowth and muscle progenitor differentiation, to critically regulate regeneration in young and aged skeletal muscle. Methods: Young C57BL/6 mice (12-14 weeks old) and old C57BL/6 mice (24-25 months old) were subjected to a previously validated model of muscle cryoinjury (n=5-6/group) to induce regeneration. The tibialis anterior muscle was injured using direct contact with a metal probe cooled in dry ice. Quantifications of cross sectional area (CSA) of myofibers were performed across 10 photos/sample by blinded reviewer via ImageJ. Western blots were performed from whole TA muscle lysates for quantification of VEGFA with GAPDH for normalization. To evaluate the role of VEGF in muscle regeneration, we performed a similar and separate series of experiments using VEGFlo (Jackson Labs Stock No. 027315) mice, which exhibit a systemic, 75% decrease in VEGFA activity, and littermate controls. In addition, ML228, a hypoxia signaling activator known to increase VEGFA levels, was injected into both young and old mice, as well as VEGFlo and littermate controls, to determine whether VEGFA augmentation promotes muscle regeneration in aging. Results: Old mice demonstrated markedly impaired ability of regenerating myofiber CSA on days post injury (DPI) 5 in comparison to young mice (Young vs. Old: 526 vs. 373µm2, p<.001) and DPI 10 (Y v. O: 1250 vs. 833µm2, p<.001). VEGFA protein levels were markedly reduced in the gastrocnemius of old mice in comparison to young. Similarly, VEGFlo mice exhibited significantly smaller CSA of regenerating fibers as compared to littermate controls (WT vs. lo: 541 vs. 238µm2, p=.0011). Pharmacologic augmentation of VEGFA, using ML228, improved skeletal muscle regeneration in both old mice and VEGFlo mice. (WT vs. lo: CSA 757 vs. 662µm2, p=.387). Conclusions: Muscle regeneration declines with with aging, in a fashion correlated to loss of VEGFA levels within skeletal muscle. Systemic loss of VEGFA is further correlated with poor muscle regeneration in genetically modified mice. Furthermore, exogenous activation of hypoxic signaling/VEGFA reverses this reduced capacity for muscle regeneration in old and VEGFlo mice. Supplementation of VEGFA could represent a therapeutic target in aging patients with skeletal muscle loss.

Effects of vascular endothelial growth factor supplementation and alginate embedding on human oocyte maturation in vitro

Objective: To evaluate whether the use of vascular endothelial growth factor (VEGF) with alginate increases oocyte maturation following in vitro maturation.Methods: This experimental study was performed on 150 immature oocytes (germinal vesicle oocytes) from females who were candidates for assisted reproductive technology. The germinal vesicle oocytes were randomly placed in the control, alginate, and VEGF plus alginate groups. The basic culture medium for oocytes culture (tissue culture medium 199, follicle-stimulating hormone 0.075 IU/mL, and fetal bovine serum 10%) was used in the control, alginate, and VEGF plus alginate groups. For the treatment groups (alginate, and VEGF plus alginate groups), alginate (8%) and VEGF (5 ng/mL) were added to the basic culture medium. After culture, immature oocytes were considered as oocytes unchanged in the nucleus whereas oocytes with a polar body were considered as mature oocytes (metaphase II stage). The mature oocytes in each group were fertilized by intracytoplasmic sperm injection and formed embryos were evaluated by reverse microscope.Results: The oocyte maturation rate (metaphase II) significantly (P < 0.05) increased in the alginate plus VEGF group as compared with the alginate alone and control groups during in vitro maturation. On day 2, the cleavage rates were significantly different in the matured oocytes between the treatment groups and the control group. The percentage of the two-cell stage, four-cell stage and eight-cell embryos was significantly higher in the treatment groups compared with the control group (P <0.05).Conclusions: Supplementation of VEGF with alginate can improve oocyte maturation in culture media. VEGF with alginate may promote the quality of nuclear and cytoplasmic maturation of human oocytes in vitro.

Ciclopirox olamine promotes the angiogenic response of endothelial cells and mesenchymal stem cells.

Prolyl hydroxylase inhibitors (PHIs) are promising compounds to promote angiogenesis by stabilizing hypoxia-inducible factor-1α (HIF-1α), a master regulator of angiogenesis. Increased HIF-1α presence induces expression of proangiogenic genes such as vascular endothelial growth factor (VEGF). We investigated the pharmacological induction of hypoxia via the PHI ciclopirox olamine (CPX) as angiogenesis strategy on human dermal microvascular endothelial cell (hd-mvEC) spheroids directly and indirectly via activating human mesenchymal stem cells (hMSCs). HMSCs were isolated from bone marrow and hd-mvECs from foreskin biopsies. MSC-conditioned medium after CPX stimulation (MSC-CM CPX) was analyzed by VEGF ELISA and Proteome Profiler™ Human Angiogenesis Array. Direct stimulation with CPX and indirect stimulation via MSC-CM CPX were compared in sprouting assays of hd-mvEC spheroids. Direct stimulation with CPX significantly increased sprouting of hd-mvEC spheroids. MSC-CM CPX also induced sprouting from hd-mvEC spheroids, which was mediated by angiogenic VEGF and other proangiogenic factors that had been produced by stimulated hMSCs. The stimulation with CPX increased the proangiogenic response of hd-mvECs and hMSCs. The direct stimulation of hd-mvECs with CPX has the potential to replace external VEGF supplementation. Thus, CPX can induce angiogenesis in ECs even in the absence of auxiliary cells demonstrating a promising proangiogenic approach.

Patient-Specific iPSC Model of a Genetic Vascular Dementia Syndrome Reveals Failure of Mural Cells to Stabilize Capillary Structures.

SummaryCADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is the most common form of genetic stroke and vascular dementia syndrome resulting from mutations in NOTCH3. To elucidate molecular mechanisms of the condition and identify drug targets, we established a patient-specific induced pluripotent stem cell (iPSC) model and demonstrated for the first time a failure of the patient iPSC-derived vascular mural cells (iPSC-MCs) in engaging and stabilizing endothelial capillary structures. The patient iPSC-MCs had reduced platelet-derived growth factor receptor β, decreased secretion of the angiogenic factor vascular endothelial growth factor (VEGF), were highly susceptible to apoptotic insults, and could induce apoptosis of adjacent endothelial cells. Supplementation of VEGF significantly rescued the capillary destabilization. Small interfering RNA knockdown of NOTCH3 in iPSC-MCs revealed a gain-of-function mechanism for the mutant NOTCH3. These disease mechanisms likely delay brain repair after stroke in CADASIL, contributing to the brain hypoperfusion and dementia in this condition, and will help to identify potential drug targets.

Blue-violet light decreases VEGFa production in an in vitro model of AMD.

Blue light is an identified risk factor for age-related macular degeneration (AMD). The production of vascular endothelial growth factor (VEGF), leading to neovascularization, is a major complication of the wet form of this disease. We investigated how blue light affects VEGF expression and secretion using A2E-loaded retinal pigment epithelium (RPE) cells, a cell model of AMD. Incubation of RPE cells with A2E resulted in a significant increase in VEGF mRNA and, intracellular and secreted VEGF protein levels, but not mRNA levels of VEGFR1 or VEGFR2. Blue light exposure of A2E-loaded RPE cells resulted in a decrease in VEGF mRNA and protein levels, but an increase in VEGFR1 levels. The toxicity of 440 nm light on A2E-loaded RPE cells was enhanced by VEGF supplementation. Our results suggest that age-related A2E accumulation may result in VEGF synthesis and release. This synthesis of VEGF, which enhances blue light toxicity for the RPE cells, is itself suppressed by blue light. Anti-VEGF therapy may therefore improve RPE survival in AMD.

Embryotropic effects of vascular endothelial growth factor on porcine embryos produced by in vitro fertilization

Current research suggests that supplementing in vitro culture (IVC) media with vascular endothelial growth factor (VEGF) may have beneficial effects on the development of porcine embryos in vitro. However, the molecular signaling mechanisms underlying this effect are unclear. Therefore, we aimed to investigate the effects of VEGF on molecular signaling events during in vitro embryonic development of porcine embryos. Porcine oocytes matured in vitro were fertilized, and the resultant zygotes were cultured with 5 ng/mL of VEGF supplemented with or without fetal bovine serum from day 4 till day 7. Without VEGF and/or FBS served as the control group. Real-time quantitative PCR was used to detect expression patterns of apoptosis- and oxidative stress-related genes in day 7 blastocysts (BLs). Early-stage apoptosis was detected by annexin-V assays in day 2 and day 7 embryos. We found that the addition of VEGF throughout the culture period with or without FBS supplementation significantly improved embryo survival and development. Supplementation with VEGF in the IVC medium significantly increased early BL formation (p < 0.05), although addition of FBS on day 4 significantly increased hatched BL formation (p < 0.05) regardless of VEGF supplementation. However, supplementation of media with both VEGF and FBS increased the formation of expanded BLs synergistically. The average total cell numbers per BL were significantly (p < 0.05) higher in embryos supplemented with VEGF and FBS than in those supplemented with either VEGF or FBS alone. We also found that accumulation of reactive oxygen species in VEGF-treated embryos was significantly lower (p < 0.05) than that in untreated embryos. The mRNA levels of caspase-3 were significantly lower (p < 0.05), and those of Bcl-2 and Nrf-2 were significantly higher (p < 0.05) in embryos grown in VEGF-supplemented media than in embryos grown in non-supplemented media. Furthermore, on day 2, the numbers of viable embryos (44.06 ± 3.94%) and blastomeres (67.18 ± 3.60%) were significantly higher (p < 0.05), and the numbers of early apoptotic embryos (55.94 ± 3.94) and blastomeres (23.23 ± 4.22) were significantly lower (p < 0.05) in VEGF-treated BLs than in controls. Furthermore, the numbers of early apoptotic cells in BLs on day 7 were also significantly lower (p < 0.05) in VEGF-treated BLs than in controls. Overall, our results indicate that supplementing IVC media with VEGF during in vitro culture of porcine embryos increases their developmental potential.

VEGF Triggers the Activation of Cofilin and the Arp2/3 Complex within the Growth Cone.

A crucial neuronal structure for the development and regeneration of neuronal networks is the axonal growth cone. Affected by different guidance cues, it grows in a predetermined direction to reach its final destination. One of those cues is the vascular endothelial growth factor (VEGF), which was identified as a positive effector for growth cone movement. These positive effects are mainly mediated by a reorganization of the actin network. This study shows that VEGF triggers a tight colocalization of cofilin and the Arp2/3 complex to the actin cytoskeleton within chicken dorsal root ganglia (DRG). Live cell imaging after microinjection of GFP (green fluorescent protein)-cofilin and RFP (red fluorescent protein)-LifeAct revealed that both labeled proteins rapidly redistributed within growth cones, and showed a congruent distribution pattern after VEGF supplementation. Disruption of signaling upstream of cofilin via blocking LIM-kinase (LIMK) activity resulted in growth cones displaying regressive growth behavior. Microinjection of GFP-p16b (a subunit of the Arp2/3 complex) and RFP-LifeAct revealed that both proteins redistributed into lamellipodia of the growth cone within minutes after VEGF stimulation. Disruption of the signaling to the Arp2/3 complex in the presence of VEGF by inhibition of N-WASP (neuronal Wiskott–Aldrich–Scott protein) caused retraction of growth cones. Hence, cofilin and the Arp2/3 complex appear to be downstream effector proteins of VEGF signaling to the actin cytoskeleton of DRG growth cones. Our data suggest that VEGF simultaneously affects different pathways for signaling to the actin cytoskeleton, since activation of cofilin occurs via inhibition of LIMK, whereas activation of Arp2/3 is achieved by stimulation of N-WASP.

Multifactorial Optimizations for Directing Endothelial Fate from Stem Cells.

Embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells are attractive in vitro models of vascular development, therapeutic angiogenesis, and tissue engineering. However, distinct ESC and iPS cell lines respond differentially to the same microenvironmental factors. Developing improved/optimized differentiation methodologies tailored/applicable in a number of distinct iPS and ESC lines remains a challenge in the field. Currently published methods for deriving endothelial cells (EC) robustly generate high numbers of endothlelial progenitor cells (EPC) within a week, but their maturation to definitive EC is much more difficult, taking up to 2 months and requiring additional purification. Therefore, we set out to examine combinations/levels of putative EC induction factors—utilizing our stage-specific chemically-defined derivation methodology in 4 ESC lines including: kinetics, cell seeding density, matrix signaling, as well as medium treatment with vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). The results indicate that temporal development in both early and late stages is the most significant factor generating the desired cells. The generation of early Flk-1+/KDR+ vascular progenitor cells (VPC) from pluripotent ESC is directed predominantly by high cell seeding density and matrix signaling from fibronectin, while VEGF supplementation was NOT statistically significant in more than one cell line, especially with fibronectin matrix which sequesters autocrine VEGF production by the differentiating stem cells. Although some groups have shown that the GSK3-kinase inhibitor (CHIR) can facilitate EPC fate, it hindered the generation of KDR+ cells in our preoptimized medium formulations. The methods summarized here significantly increased the production of mature vascular endothelial (VE)-cadherin+ EC, with up to 93% and 57% purity from mouse and human ESC, respectively, before VE-cadherin+ EC purification.

MEK/ERK signaling is involved in the role of VEGF and IGF1 in cardiomyocyte differentiation of mouse adipose tissue-derived stromal cells

BackgroundWe found that mouse brown adipose tissue-derived stromal cells (BATDCs), but not white adipose tissue-derived stromal cells (WATDCs), could spontaneously differentiate into cardiomyocyte-like cells in a simple culture medium. This study would find out some critical trophic factors that were responsible for such difference in differentiation, and further determine the involved signaling pathway. Methods and resultsThe cardiomyocyte differentiation capacity of cells was identified by morphological observations, immunofluorescence staining, and evaluation of expression of cardiomyocyte specific markers. The amount of vascular endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF1) secreted by cells was determined by ELISA analysis. Results indicated that BATDCs secreted higher levels of VEGF and IGF1 than WATDCs. Supplementation of BATDCs with antibodies against VEGF receptor Flt-1 or IGF1 receptor Igf-1rα significantly suppressed the cardiac differentiation capacity of the cells. Additionally, anti-Flt-1 and anti-Igf-1rα antibodies decreased phosphorylation of ERK1/2 in BATDCs. Inhibition of MEK/ERK activity by the inhibitor PD0325901 or by RNA interference blunted the cardiac differentiation of BATDCs. Loading recombinant VEGF and IGF1, or transfecting their expression vectors into WATDCs, promoted cardiac differentiation of the cells. Preincubation with PD0325901, before VEGF and IGF1 supplementation or vector transfections, blocked the stimulation of cardiac differentiation in WATDCs. ConclusionsThese findings indicated that VEGF and IGF1 were critical factors for the spontaneous cardiac differentiation of BATDCs, and MEK/ERK signaling was involved in the role of VEGF and IGF1. VEGF and IGF1 could be used to promote the development of cardiomyocyte phenotype in WATDCs.

Therapeutic angiogenesis by vascular endothelial growth factor supplementation for treatment of renal disease.

Vascular endothelial growth factors (VEGFs) influence renal function through angiogenesis, with VEGF-A being the most potent inducer of vascular formation. In the normal glomerulus, tight homeostatic balance is maintained between the levels of VEGF-A isoforms produced by podocyte cells, and the VEGF receptors (VEGFRs) expressed by glomerular endothelial, mesangial, and podocyte cells. Renal disease occurs when this homeostatic balance is lost, manifesting in the abnormal autocrine and paracrine VEGF-A/VEGFR signaling, ultrastructural glomerular and tubular damage, and impaired filtration. Preclinical disease models of ischemic renal injury, including acute ischemia/reperfusion, thrombotic microangiopathy, and chronic renovascular disease, treated with exogenous VEGF supplementation demonstrated therapeutic efficacy. These results suggest a therapeutic VEGF-A paracrine effect on endothelial cells in the context of acute or chronic obstructive ischemia. Conversely, renal dysfunction in diabetic nephropathy appears to occur through an upregulated VEGF autocrine effect on podocyte cells, which is exacerbated by hyperglycemia. Therefore, VEGF supplementation therapy may be contraindicated for treatment of diabetic nephropathy, but specific results will depend on dose and on the specific site of VEGF delivery. A drug delivery system that demonstrates cell specificity for glomerular or peritubular capillaries could be employed to restore balance to VEGF-A/VEGFR2 signaling, and by doing so, prevent the progression to end-stage renal disease. The review discusses the preclinical data available for VEGF supplementation therapy in models of renal disease.

Human endothelial and foetal femur-derived stem cell co-cultures modulate osteogenesis and angiogenesis.

BackgroundA dynamic vasculature is a prerequisite for bone formation where the interaction of bone cells and endothelial cells is essential for both the development and the healing process of bone. Enhanced understanding of the specific mediators involved in bone cell and endothelial cell interactions offers new avenues for skeletal regenerative applications. This study has investigated the osteogenic and angiogenic potential of co-cultures of human foetal diaphyseal or epiphyseal cells with human umbilical vein endothelial cells (HUVEC) in the presence and absence of vascular endothelial growth factor (VEGF) supplementation.MethodsEarly osteogenic activities of the co-cultures (±VEGF) were assessed by alkaline phosphatase (ALP) activity. Osteogenic and angiogenic gene expression was measured using quantitative polymerase chain reaction. An ex vivo organotypic embryonic chick (E11) femur culture model was used to determine the osteogenic effects of VEGF as determined using micro-computed tomography (μCT) and Alcian blue/Sirius red histochemistry and immunocytochemistry for expression of CD31.ResultsALP activity and gene expression of ALP and Type-1 collagen was enhanced in foetal skeletal/HUVECs co-cultures. In foetal diaphyseal/HUVECs co-cultures, VEGF reduced the levels of ALP activity and displayed a negligible effect on von Willebrand factor (vWF) and VEGF gene expression. In contrast, VEGF supplementation was observed to significantly increase FLT-1 and KDR gene expression in co-cultures with modulation of expression enhanced, compared to VEGF skeletal monocultures. In the organotypic chick model, addition of VEGF significantly enhanced bone formation, which coincided with elevated levels of CD31-positive cells in the mid-diaphyseal region of the femurs.ConclusionThese studies demonstrate a differential skeletal response of early foetal skeletal cells, when co-cultured with endothelial cells and the potential of co-culture models for bone repair. The differential effect of VEGF supplementation on markers of angiogenesis and osteogenesis in co-cultures and organ cultures, demonstrate the importance of the intricate temporal coordination of osteogenic and angiogenic processes during bone formation and implications therein for effective approaches to bone regenerative therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0270-3) contains supplementary material, which is available to authorized users.

Role of VEGF and VEGFR2 Receptor in Reversal of ALS-CSF Induced Degeneration of NSC-34 Motor Neuron Cell Line.

Vascular endothelial growth factor (VEGF), the well-known angiogenic factor is both neurotrophic and neuroprotective. Altered VEGF signalling is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), a fatal degenerative disease of motor neurons. We have shown earlier that VEGF protects NSC-34 motor neuronal cell line, when exposed to cerebrospinal fluid (CSF) from sporadic ALS patients (ALS-CSF). Here, we have investigated the consequences of ALS-CSF and VEGF supplementation on the VEGFR2 receptor and endogenous VEGF expression. ALS-CSF caused significant down-regulation of VEGFR2 as well as the Calbindin-D28K levels, but not endogenous VEGF. Exogenous supplementation restored the depletion of VEGFR2 and Calbindin-D28K with a concomitant up-regulation of endogenous VEGF. The up-regulated caspase 3 in the ALS-CSF group was reinstated to basal levels along with a significant reduction in the number of TUNEL-positive cells. Electron photomicrographs of ALS-CSF-exposed cells divulged presence of cytoplasmic vacuoles alongside severe damage to organelles like mitochondria, endoplasmic reticulum, etc. Substantial recovery of most of the damaged organelles was noted in response to VEGF supplementation. While the enhancement in endogenous VEGF levels highlights the autocrine functions, the up-regulation of VEGFR2 receptor emphasizes the paracrine functions of VEGF in modulating its neuroprotective effect against ALS-CSF. The revival of cellular organellar structure, increased calbindin expression and enhanced survival in response to VEGF supplementation consolidates the opinion that VEGF indeed has a therapeutic potential in sporadic ALS.

CREG Promotes the Proliferation of Human Umbilical Vein Endothelial Cells through the ERK/Cyclin E Signaling Pathway

Cellular repressor of E1A-stimulated genes (CREG) is a recently discovered secreted glycoprotein involved in homeostatic modulation. We previously reported that CREG is abundantly expressed in the adult vascular endothelium and dramatically downregulated in atherosclerotic lesions. In addition, CREG participates in the regulation of apoptosis, inflammation and wound healing of vascular endothelial cells. In the present study, we attempted to investigate the effect of CREG on the proliferation of vascular endothelial cells and to decipher the underlying molecular mechanisms. Overexpression of CREG in human umbilical vein endothelial cells (HUVEC) was obtained by infection with adenovirus carrying CREG. HUVEC proliferation was investigated by flow cytometry and 5-bromo-2′-deoxy-uridine (BrdU) incorporation assays. The expressions of cyclins, cyclin-dependent kinases and signaling molecules were also examined. In CREG-overexpressing cells, we observed a marked increase in the proportion of the S and G2 population and a decrease in the G0/G1 phase population. The number of BrdU positively-stained cells also increased, obviously. Furthermore, silencing of CREG expression by specific short hairpin RNA effectively inhibited the proliferation of human umbilical vein endothelial cells (HUVEC). CREG overexpression induced the expression of cyclin E in both protein and mRNA levels to regulate cell cycle progression. Further investigation using inhibitor blocking analysis identified that ERK activation mediated the CREG modulation of the proliferation and cyclin E expression in HUVEC. In addition, blocking vascular endothelial growth factor (VEGF) in CREG-overexpressed HUVEC and supplementation of VEGF in CREG knocked-down HUVEC identified that the pro-proliferative effect of CREG was partially mediated by VEGF-induced ERK/cyclin E activation. These results suggest a novel role of CREG to promote HUVEC proliferation through the ERK/cyclin E signaling pathway.

Role of salivary vascular endothelial growth factor (VEGF) in palatal mucosal wound healing

The mucosa of alimentary tract heals more rapidly than cutaneous wounds. The underlying mechanisms of this enhanced healing have not been completely elucidated. Constant exposure to salivary growth factors has been shown to play a critical role in mucosal homeostasis and tissue repair. Angiogenesis also has an essential role in successful wound repair. One of the main angiogenic growth factors, vascular endothelial growth factor (VEGF), has a pleiotropic role in tissue repair via neovascularization, reepithelialization, and regulation of extracellular matrix. We have previously reported a critical role for salivary VEGF in bowel adaptation after small bowel resection. We hypothesize that salivary VEGF is an essential stimulus for oral mucosal tissue repair, and use the murine palatal wound model to test our hypothesis. In a loss-of-function experiment, we removed the primary source of VEGF production through selective submandibular gland (SMG) sialoadenectomy in a murine model and observed the effects on wound closure and neovascularization. We then performed a selective loss-of-function experiment using the protein VEGF-Trap to inhibit salivary VEGF. In a gain-of-function experiment, we supplemented oral VEGF following SMG sialoadenectomy. After SMG sialoadenectomy, there was significant reduction in salivary VEGF level, wound closure, and vessel density. Lower levels of salivary VEGF were correlated with impaired neovascularization and reepithelialization. The selective blockade of VEGF using VEGF-Trap resulted in a similar impairment in wound healing and neovascularization. The sole supplementation of oral VEGF after SMG sialoadenectomy rescued the impaired wound healing phenotype and restored neovascularization to normal levels. These data show a novel role for salivary-VEGF in mucosal wound healing, and provide a basis for the development of novel therapeutics aimed at augmenting wound repair of the oral mucosa, as well as wounds at other sites in the alimentary tract.

Reduction of connexin43 in human endothelial progenitor cells impairs the angiogenic potential

Our previous work showed that arsenic trioxide down-regulated Cx43 and attenuated the angiogenic potential of human late endothelial progenitor cells (EPC). However, the relation between Cx43 and angiogenic activity of the EPC remained unclear. In the study, human late EPC were treated with siRNA specific to Cx43 (Cx43siRNA). The expression profiles as well as activity of the treated cells were examined. In parallel, the angiogenic potential of human EPC treated with Cx43siRNA was evaluated using murine hind limb ischemic model. The results showed that, in the EPC treated with Cx43siRNA, the activity of migration, proliferation, and angiogenic potential were attenuated, accompanied by reduction in vascular endothelial growth factor (VEGF) expression. In hind limb ischemia mice, EPC treated with Cx43siRNA lost the therapeutic angiogenic potential. VEGF supplementation partially recovered the activity impaired by Cx43 down-regulation. In conclusion, reduced Cx43 expression per se in the EPC causes decreased expression of VEGF and impaired angiogenic potential of the cells. Prevention of Cx43 reduction is a potential target to maintain the angiogenic potential of the EPC.

Exogenous vascular endothelial growth factor supplementation can restore the podocyte barrier‐forming capacity disrupted by sera of preeclamptic women

To determine whether exogenous vascular endothelial growth factor (VEGF) supplementation to sera from women with preeclampsia can restore the podocyte barrier-forming capacity disrupted after stimulation with sera from these women. Enzyme-linked immunosorbent assays (ELISAs) for sFlt-1 (soluble VEGF receptor 1) and VEGF were performed in the sera of preeclamptic and healthy pregnant women. VEGF-enriched preeclamptic serum was used to evaluate the capacity of such sera to restore the diminished podocyte barrier capacity secondary to stimulation with untreated preeclamptic sera. Two groups, preeclampsia (n=15) and healthy (n=15) pregnant women, were established. Median serum levels (pg/mL) of sFlt-1 and VEGF were significantly different between groups: 3987 versus 1432 and 6 versus 15.6 (P<0.001). Altered resistance values of podocytes stimulated with sera from women with preeclampsia were recovered after exogenous VEGF supplementation. The mean resistance value of cultured podocytes, an indicator of the integrity of the cytoskeleton and the capacity of these cells to form layers, is altered in women with preeclampsia. High levels of sFlt-1, an inhibitor of VEGF observed in high concentrations in the serum of these patients, could be the underlying cause of this alteration. We provide evidence that exogenous VEGF supplementation of the sera of these women could recover this indicator.

Salivary VEGF Plays an Essential Role in Oral Mucosal Wound Healing

Introduction: Palatal mucosal wound healing occurs more rapidly than in cutaneous wounds. Vascular endothelial growth factor (VEGF) induced angiogenesis is critical for cutaneous wound healing. We have previously demonstrated 1) salivary glands produce high levels of VEGF and 2) an essential role for salivary VEGF (salVEGF) in the intestinal response to injury. This has led us to hypothesize that salVEGF is critical in oral mucosal wound healing and neovascularization. to test this hypothesis, we performed a loss-of-function experiment by selectively inhibiting salVEGF using a novel adenoviral construct (AdVegfTrap) and a gain-of-function experiment by supplementing oral VEGF following submandibular gland (SMG) resection. Methods: In the loss-of-function experiment, C57/BL6 mice underwent SMG duct cannulation and were treated with 1x108 PFU AdVegfTrap, AdLacZ or PBS (n=5/group). 1.5 mm palatal mucosal wounds were created. in the gain-of-function experiment, C57/BL6 mice underwent SMG sialoadenectomy (n=4) or sham operation (n=5). 1.5 mm palatal wounds were created and oral VEGF protein was supplemented. AdVegfTrap was not used due to anticipated consumption of the orally replaced VEGF. in both experiments, salVEGF protein levels were quantified by ELISA. Wounds were harvested at 3 days and analyzed for epithelial gap and vessel density. Data were expressed as mean+SEM. Results: Retrograde SMG duct injection of AdLacZ results in 100% transduction efficiency confirming effective transgene delivery. SMG duct cannulation, SMG administration of AdLacZ and sham operation controls do not result in significantly decreased of salVEGF (PBS513pg/ml±141 vs AdLacZ353pg/ml±56 vs sham472pg/ml±40 vs control514pg/ml±89;p=NS). Administration of AdVegfTrap results in depletion of salVEGF comparable to SMG sialoadenectomy (AdVegfTrap122pg/ml±42 vs SMG resection136pg/ml±15.9;p=NS). Selective inhibition of salVEGF by AdVegfTrap impairs wound closure (AdVegfTrap947μm±22 vs AdLacZ411μm±21 vs PBS355μm±31;p<0.001) and vessel density (AdVegfTrap15.95caps/hpf±0.83 vs AdLacZ30.87caps/hpf±1.1 vs PBS30.5caps/hpf±0.95;p<0.001). Oral replacement of VEGF in SMG resected mice rescues the impaired reepithelialization and neovascularization phenotype (SMG with oral VEGF194μm±43 vs sham350μm±46,p=NS; SMG with oral VEGF24.0caps/hpf±1.4 vs sham18.6caps/hpf±2.1,p=NS). Conclusions: Selective inhibition of salVEGF significantly impairs palatal reepithelialization and neovascularization. Oral supplementation of VEGF rescues the impaired wound healing phenotype in SMG sialoadenectomized mice. Taken together, these findings suggest that salVEGF is an essential growth factor in oral mucosal wound healing which may be a surrogate for intestinal wound healing. the palatal wound model may be a valuable tool in understanding the critical role of salVEGF in the alimentary tract response to injury.