Vascular Endothelial Growth Factor Expression In Endothelial Cells Research Articles

A two-pronged approach to regulate the behaviors of ECs and SMCs by the dual targeting-nanoparticles

Inhibiting vascular restenosis remains a tricky challenge for the postoperative development of cardiovascular interventional therapy. The ideal approaches should activate endothelial cells (ECs) and restrain smooth muscle cells (SMCs), however, they are commonly contradictory. Herein, a strategy was developed for synchronizing ECs promotion and SMCs inhibition by codelivery DNA and siRNA for combination therapy. Thus, an easy and efficient strategy integrated dual-superiorities of precise targeting and dual therapeutic genes to precisely regulate the behaviors of ECs and SMCs. The ECs-targeting REDV peptide and SMCs-targeting VAPG peptide grafted anionic polymers were used to surface-functionalize the delivery nanoplatforms for vascular endothelial growth factor (VEGF) plasmids and ERK2 siRNA delivery, respectively. The dual targeting-nanoparticles were prepared by physical mixing method, and their outstanding advantages were confirmed by the co-culture experiments. In comparison with single targeting-nanoparticles and dual non-targeting-nanoparticles, the dual targeting-nanoparticles simultaneously enhanced ECs proliferation/migration and restrained SMCs proliferation/migration. Moreover, the dual targeting-nanoparticles group manifested the highest VEGF expression in ECs and the lowest ERK2 expression in SMCs. In summary, the two-pronged strategy with dual targeting-nanoparticles provides a valuable cornerstone for synchronizing ECs promotion and SMCs inhibition.

Regulation of VEGF-A Expression in Endothelial Cells by Transcriptional Gene Activation or Transcriptional Gene Silencing: Analysis of Genome Wide Transcriptional Response

Vascular endothelial growth factor (VEGF-A) is an important gene in many diseases, such as cancer and cardiovascular diseases. We investigated changes in genome-wide gene expression patterns in murine endothelial cells when the expression of VEGF-A was epigenetically modulated using promoter targeted small hairpin RNAs (shRNAs). Murine endothelial cells were transduced with lentiviral vectors expressing shRNAs that are complementary to the gene promoter. Gene expression array experiments were conducted to investigate genome-wide mRNA expression changes caused by up- and downregulating VEGF-A. There were several hundreds of differentially expressed genes according to the applied statistical criteria. We noticed that the effects of downregulating VEGF-A were much more wide-spread than the effects of VEGF-A upregulation. Our in silico analysis revealed that a number of different biological processes are altered due to epigenetic effects on VEGF-A expression. One of the main regulators of VEGF-A mediated transcriptional response was found to be transcription factor ATF-4. This is the first study showing the transcriptional response to epigenetic modification of VEGF-A expression in endothelial cells. Epigenetic gene regulation represents a natural gene regulatory mechanism and these results reveal previously unknown implications of VEGF-A regulation in endothelial cells.

Shock wave treatment enhances endothelial proliferation via autocrine vascular endothelial growth factor.

Extracorporeal cardiac shock wave (SW) therapy is an effective, safe, and non-invasive therapeutic strategy for severe coronary artery disease. Shock wave therapy might affect cardiac tissues because of its ability to promote angiogenesis. In this report, we investigated if the up-regulation of vascular endothelial growth factor (VEGF) by SW therapy is involved in cell proliferation in cultured endothelial cells. After human umbilical vein endothelial cells were treated with SW, the expression and secretion of VEGF as well as cell proliferation were analyzed. We also determined the mechanism underlying SW-induced the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) using western blotting. Our results demonstrated that SW treatment induced VEGF expression in endothelial cells in a hypoxia-inducible factor 1-independent manner. Up-regulation of VEGF expression led to an increase in its concentration in the cultured medium. The autocrine VEGF in the medium activated the ERK MAPK signaling, which in turn enhanced cell proliferation. Therefore, we concluded that VEGF mediates SW application-induced endothelial cell proliferation in a cell-autonomous manner.

Expression of Vascular Endothelial Growth Factor (VEGF) in Macrophages, Fibroblasts, and Endothelial Cells in Pterygium Treated with 5-Fluorouracil

Purpose: To evaluate the VEGF expression in macrophages, fibroblasts, and endothelial cells from pterygium before and after 5-fluorouracil (5-FU) exposure.Methods: 68 excised pterygia (42 primary and 26 recurrent) were studied by immunohistochemistry, to analyze its expression in cells from pterygium and normal conjunctiva. VEGF expression was evaluated before and after a 5-FU subconjunctival injection 15 days prior to surgery.Results: Expression in macrophages was moderate in normal conjunctiva and low to moderate in pterygium tissues. In fibroblasts, it was negative or low in both tissues. Pterygium had a higher proportion of high VEGF expression in endothelial cells compared to normal tissue (p < 0.05). 5-FU did not have any influence on expression.Conclusion: VEGF expression in pterygium macrophages, fibroblasts, and endothelial cells was low and similar to that normal in conjunctival tissue. High VEGF expression was found in pterygium endothelial cells compared to normal conjunctiva. 5-FU does not impact VEGF expression.

Regulation of Vascular Endothelial Growth Factor Expression by Extra Domain B Segment of Fibronectin in Endothelial Cells

Diabetic retinopathy entails proliferation of vascular endothelial cells (ECs) and unregulated angiogenesis. We have previously shown that ECs increase the expression of an embryonic variant of fibronectin (FN), called extra domain-B FN (ED-B FN) in response to high glucose. We also showed that ED-B FN regulates EC tube morphogenesis, possibly through vascular endothelial growth factor (VEGF). In the present study, we have attempted to decipher the mechanisms by which ED-B FN may modulate EC phenotype. We hypothesized that ED-B FN regulates VEGF expression in ECs through interaction with selected integrin receptors. To test this hypothesis, we first cultured ECs in high levels of glucose to investigate for any alteration. We then used integrin-specific matrix mimetic peptides, neutralizing antibodies, and RNAi to identify the integrin(s) involved in VEGF expression. Finally, we used an animal model of diabetes to study whether these in vitro mechanisms also take place in the retina. Our results show that exposure of ECs to high levels of glucose increased VEGF expression. ED-B FN mediated this increase since knockdown of ED-B FN completely prevented glucose-induced VEGF expression. We then identified β1 integrin as the essential receptor involved in high glucose-induced VEGF expression. We also showed that diabetes increased β1 integrin and VEGF expression in the retina, which normalized upon ED-B knockdown. These findings showed that high levels of glucose in diabetes increased VEGF expression in ECs through ED-B FN and β1 integrin interaction. These results provide novel mechanistic basis of increased VEGF expression in diabetes.

Morphine and angiogenesis in hypertensive animals.

In a recent published paper, Pourshanazari et al found that morphine enhanced angiogenesis in murine matrigel method in sham and two-kidney one-clip (2K-1C) hypertensive animals and they concluded that this effect is via NO pathway (1). Morphine acts both directly and indirectly on angiogenesis, however, the results are contradictory. It seems that the different effect of morphine on angiogenesis process depends on the low or high dose and acute or chronic morphine administration. It is indicated that chronic morphine treatment in wound repair resulted in a significant decrease in the expression of HIF-1α and reduced vascular endothelial growth factor (VEGF) synthesis (2). Morphine increases systemic oxidative stress, impairs mobilization of endothelial progenitor cells (3) and inhibits hypoxiainduced VEGF expression in endothelial cells and cardiac myocytes in ischemic myocardium (4). Morphine also suppresses tumor angiogenesis through a HIF-1alpha/p38MAPK pathway (5). In contrast, it is able to stimulate vascular endothelial cell proliferation in vitro through mitogen-activated protein kinase (MAPK) pathway (6). Topical use of opioids accelerate healing by up-regulating both endothelial and inducible nitric oxide synthase and the vascular endothelial-derived growth factor receptor Flk1 in the wounds (7). Thus, it seems that the effect of morphine on angiogenesis depends on the type of experimental model as well. The authors also concluded that morphine enhanced angiogenesis in sham and 2K-1C animals via NO pathway and they showed that angiogenesis in hypertensive animals is much higher than sham group. Hypertensive subjects have reduced NO bioavailability due to several mechanisms (8) and in that study, morphine increased serum NO concentration in 2K-1C animals which approached normal level. Thus, it seems that the increased serum NO level could not completely explain enhanced angiogenesis in morphine-treated hypertensive animals. On the other hand, there is a relationship between NO and angiotensin II (Ang II). It was shown that high Ang II level decreases NO bioavailability by promoting oxidative stress (9). Thus, morphine probably reduced plasma renin activity and Ang II levels which results in increased NO level. More studies need to clarify the exact role and mechanism of angiogenesis during morphine administration.

Angiogenesis in the regenerating tail of the leopard gecko, Eublepharis macularius

Angiogenesis has well documented roles in embryonic development and tumourigenesis, and is also involved in the phenomenon of tissue repair and regeneration. We investigated angiogenesis during complex multi-tissue regeneration using the leopard gecko tail as a novel model. Similar to many lizards, leopard geckos are able to naturally regenerate their tails following predation. Using various mammalian angiogenic markers (alpha-smooth muscle actin (α-SMA), vascular endothelial growth factor (VEGF), thrombospondin-1 (TSP-1), and cluster of differentiation 36 (CD36)) we characterized angiogenesis during reparative regeneration. After an initial wound healing phase, tail regeneration begins with the formation of an aggregation of proliferating cells (blastema). As the blastema increases in size new capillaries develop, lined by VEGF positive endothelial cells (ECs). Continued maturation of vessels corresponds with the recruitment of α-SMA positive pericytes, as well as ongoing VEGF expression in ECs. While TSP-1 is not expressed in ECs, it is seen transiently in cells of the blastema immediately following wound healing. CD36 is expressed in ECs of the late stage regenerate tail, presumably involved in TSP-1 mediated vessel remodelling. We demonstrate that regenerative angiogenesis forms an organized vascular network through a highly regulated process comparable to embryonic embryogenesis. Grant Funding Source: NSERC, CIHR

CXCL8/IL8 Stimulates Vascular Endothelial Growth Factor (VEGF) Expression and the Autocrine Activation of VEGFR2 in Endothelial Cells by Activating NFκB through the CBM (Carma3/Bcl10/Malt1) Complex

Vascular endothelial growth factor (VEGF) is a potent mitogen and permeability factor for endothelial cells that plays a central role in angiogenesis, vascular maintenance, inflammation, and cancer. VEGF also mediates the homeostatic adaptation to hypoxic conditions by promoting an increase in vascular density to compensate for decreased oxygenation. This process is triggered by an oxygen-sensitive transcription factor, hypoxia-inducible factor-1 (HIF1alpha), which becomes active in hypoxic tissues, leading to the synthesis and secretion of VEGF. The role of HIF1alpha in other processes that involve angiogenesis such as in inflammation is less clear. Of interest, endothelial cells not only respond to but also store and secrete VEGF, which is required for the maintenance of the integrity of the vascular system. How this intracellular pool of VEGF is regulated is still not understood. Here, we found that CXCL8/IL8, a potent proangiogenic and inflammatory chemokine, up-regulates VEGF mRNA and protein levels in endothelial cells by acting on its cognate receptor, CXCR2, and that this results in the autocrine activation of VEGFR2. Surprisingly, this process does not involve HIF1alpha but instead requires the activation of the transcription factor NFkappaB. Furthermore, we identified the components of the CBM complex, Carma3, Bcl10, and Malt1, as key mediators of the CXCL8/IL8-induced NFkappaB activation and VEGF up-regulation. Together, these findings support the existence of an NFkappaB-mediated pathway by which the proinflammatory chemokine CXCL8/IL8 controls the expression of VEGF in endothelial cells, thereby promoting the activation of VEGF receptors in an autocrine fashion.

CD40-Induced Signaling in Human Endothelial Cells Results in mTORC2- and Akt-Dependent Expression of Vascular Endothelial Growth Factor In Vitro and In Vivo

We have examined CD40-dependent signals in endothelial cells (EC) mediating the expression of vascular endothelial growth factor (VEGF) and VEGF-induced angiogenesis. We treated confluent cultures of EC with soluble CD40L (sCD40L), and by Western blot found a marked increase in the phosphorylation of Akt, 4EBP-1, and S6K1, compared with untreated cells. EC were transfected with a full-length VEGF promoter-luciferase construct and cultured in the absence or presence of rapamycin and sCD40L. We found that rapamycin, which blocks mTORC1 and mTORC2 signaling, inhibited sCD40L-mediated transactivation of VEGF. In addition, by Western blot, we found that the transfection of EC with small interfering RNA (siRNA) to rictor (to inhibit mTORC2), and not raptor (to inhibit mTORC1), inhibited sCD40L-dependent protein expression of VEGF. In additions, we found that basal levels of phosphorylated Akt as well as VEGF were increased in EC transfected with the raptor siRNA. Also, rapamycin failed to inhibit VEGF promoter activation, as well as VEGF protein expression in EC transfected with a constitutively active construct of Akt, further demonstrating that mTORC1 is not necessary for CD40- and Akt-induced expression of VEGF. Finally, we injected human CD40L-transfected fibroblasts or mock transfectants into human skin on SCID mice. We found that the injection of CD40L transfectants, but not mock cells, resulted in VEGF expression and mediated a marked angiogenesis reaction, and this response was reduced in mice treated with rapamycin. Together, these observations indicate that mTORC2 and Akt facilitate CD40-inducible expression of VEGF in EC, which is of clinical importance in tumor growth and the progression of chronic inflammatory diseases.

Implications of bevacizumab on vascular endothelial growth factor and endostatin in human choroidal neovascularisation

Aim:To evaluate the implications of intravitreal bevacizumab on proangiogenic vascular endothelial growth factor (VEGF) with regard to the endogenous angiogenesis inhibitor endostatin in human choroidal neovascularisation (CNV) secondary to age-related...

FGF Signaling Preserves the Integrity of Endothelial Adherens Junctions

Fibroblast growth factor (FGF)-activated signaling regulates an array of cellular processes ranging from embryonic development to tissue repair. A recent paper by Murakami et al. identifies a potentially important role for FGF signaling in maintenance of endothelial barrier homeostasis through the regulation of adherens junctions.

Simvastatin Stimulates Vascular Endothelial Growth Factor Production by Hypoxia-inducible Factor-1α Upregulation in Endothelial Cells

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor and plays an important pathophysiological role in the maintenance of tissue structure as well as regeneration after ischemic injury. Three-hydroxy-3methylglutaryl-CoA reductase inhibitors reduce vascular inflammation and induce angiogenesis. This study examined whether simvastatin stimulates VEGF expression in endothelial cells as well as the nature of its underlying mechanism. Simvastatin induced mRNA expression and protein secretion of VEGF in endothelial cells that were reversed by pretreatment with mevalonate and geranylgeranylpyrophosphate but not by farnesylpyrophosphate. Adenovirus-mediated expression of the dominant-negative mutant of RhoA induced VEGF mRNA and protein. Simvastatin increased hypoxia-inducible factor-1alpha (HIF-1alpha) protein level without changing its mRNA expression. Inhibition of RhoA had similar effects to simvastatin on VEGF expression. Inhibition of RhoA caused the translocation of HIF-1alpha to the nuclear fraction. Depletion of HIF-1alpha by RNA interference blocked simvastatin-induced VEGF mRNA expression. Simvastatin stimulates VEGF expression by RhoA downregulation and HIF-1alpha upregulation in endothelial cells. These data indicate a novel role for RhoA as a negative regulator of HIF-1alpha.

Modulation of angiogenic processes in cultured endothelial cells by low density lipoproteins subfractions from patients with familial hypercholesterolemia

Objective Electronegative low density lipoprotein (LDL) subfractions are cytotoxic to endothelial cells. To continue our study of homozygotic familial hypercholesterolemic (FH)-LDL, we report the effects of FH-LDL subfractions (FH-L1 to FH-L5) on the angiogenic processes in cultured endothelial cells. Methods and results Subconfluent bovine aortic endothelial cells (BAEC) were treated with LDL subfractions (20 μg/ml), and the effects on angiogenic functions, including cell proliferation, migration, apoptosis, tube formation, secretion of matrix metalloproteinases (MMPs), and vascular endothelial growth factor (VEGF) were determined. The electronegative FH-L4 and FH-L5 inhibited cell proliferation while the other FH-LDL subfractions and LDL from normocholesterolemic subjects (N-LDL) had negligible effects. Like Cu 2+ ox-LDL, FH-L5 strongly inhibited endothelial cell viability and FH-L4 had a milder effects. Similarly, FH-L4 and FH-L5 but not the other subfractions retarded cell migration, induced cell apoptosis, and perturbed tube formation by BAEC in matrigel. FH-L5 inhibited secretion of MMP-2 and MMP-9 by BAEC without affecting their endogenous levels. In contrast, FH-L5 increased the VEGF expression in endothelial cells. Conclusions Our results show for the first time that FH-L5, a circulating LDL subfraction from hypercholesterolemic patients, modulates various angiogenic processes, thereby dysregulating endothelial function in a way that may be atherogenic.

Transformation of vascular endothelial cells by a point mutation in the Tie2 gene from human intramuscular haemangioma.

Tie2, an endothelial-cell-specific receptor tyrosine kinase, collaborates with vascular endothelial growth factor (VEGF) in regulating angiogenesis and vascular maturation. Here, we report a mutation of glycine to aspartic acid at the second glycine of the GXGXXG motif of Tie2 (G833DTie2) in human intramuscular haemangiomas (IMHs) of the capillary type. Murine endothelial cells (ECs) overexpressing this G833DTie2 receptor exhibited an increase in cell proliferation at low serum concentrations and angiosarcomas developed in nude mice, whereas cells overexpressing either wild-type Tie2 or Q837HTie2 failed to elicit these responses. Furthermore, the G833DTie2 receptor increased VEGF expression in ECs. These findings provide molecular mechanisms for pathogenesis of IMH

Platelet-activating factor (PAF) induces corneal neovascularization and upregulates VEGF expression in endothelial cells.

Platelet-activating factor (PAF) is a potent proinflammatory mediator that accumulates in the cornea after injury and induces the expression of genes related to inflammation and wound healing. The current study was conducted to investigate the direct effect of PAF on corneal neovascularization and on the expression of angiogenic growth factors in vascular endothelial cells. Pellets containing carbamyl-PAF (cPAF) were implanted in corneas of wild-type or PAF-receptor (PAF-R)-knockout mice, and the progression of angiogenesis was monitored by microscope. In some experiments, mice were treated with a daily intraperitoneal injection of the PAF-R antagonist LAU8080. Migration assays of human umbilical cord vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HMVECs) were performed in a Boyden chamber after addition of various concentrations of cPAF or bovine fibroblast growth factor (FGF-2). Cell proliferation was assessed by fluorescence-binding assay in the presence of cPAF or FGF-2 for 8 days. Vascular endothelial growth factor (VEGF) and FGF-2 expression was studied by RT-PCR and Northern- and Western-blot analyses in cells stimulated with cPAF at different concentrations and for different times. Six days after cPAF pellet implantation, there were new vessels growing from the limbus to the center of the cornea. The PAF-induced neovascularization was significantly reduced in PAF-R-knockout mice and in mice treated with the PAF antagonist. cPAF added to the lower well of the Boyden chamber produced a dose-dependent migration of HUVECs and HMVECs that was inhibited in cells preincubated with LAU8080 or with a VEGF-blocking antibody. In contrast, cPAF did not stimulate proliferation of endothelial cells. cPAF induced VEGF mRNA and protein expression but not FGF-2 expression in HUVECs and HMVECs. PAF stimulates corneal neovascularization by a receptor-mediated mechanism. Induction of VEGF expression and stimulation of vascular endothelial cell migration are initial events in PAF-promoted corneal angiogenesis.

Nicotine and Cotinine Up-Regulate Vascular Endothelial Growth Factor Expression in Endothelial Cells

Cigarette smoking is an important risk factor for both vascular disease and various forms of cancer. Vascular endothelial growth factor (VEGF) is an endothelial-specific mitogen that is normally expressed only in low levels in normal arteries but may be involved in the progression of both vascular disease and cancer. Some clinical evidence suggests that cigarette smoking may increase plasma VEGF levels, but there is a lack of basic science studies investigating this possibility. We show here, using an intact porcine common carotid artery perfusion culture model, that nicotine and cotinine, the major product of nicotine metabolism, cause a significant increase in endothelial cell VEGF expression. VEGF mRNA levels were compared between groups using reverse transcriptase-polymerase chain reaction, whereas protein level changes were demonstrated with Western blotting and immunohistochemistry. Our results showed significant increases in endothelial cell VEGF mRNA and protein levels because of nicotine and cotinine at concentrations representative of plasma concentrations seen in habitual smokers. VEGF immunostaining also paralleled these results. These findings may give a clue as to the mechanisms by which nicotine and cotinine from cigarette smoking increase vascular disease progression and tumor growth and metastasis.

Morphine Sulfate Inhibits Hypoxia-induced Vascular Endothelial Growth Factor Expression in Endothelial Cells and Cardiac Myocytes

Vascular endothelial growth factor (VEGF) is an angiogenic mitogen, specific for endothelial cells. Hypoxia-induced VEGF in endothelial cells and cardiomyocytes leads to autocrine and paracrine stimulation, respectively. During myocardial ischemia, VEGF is upregulated in the endothelium and myocardium, and may mediate angiogenesis. Morphine sulfate is commonly used in pain relief for patients with acute myocardial infarction. We investigated the effect of morphine sulfate on VEGF expression in cultured endothelial cells and cardiac myocytes subjected to hypoxia. Enzyme-linked immunosorbent assays showed that morphine sulfate significantly inhibited hypoxia-induced VEGF expression in mouse heart microvascular endothelial cells (SMHEC4), primary cultures of human umbilical vein endothelial cells (HUVECs) and in primary cultures of rat cardiac myocytes (P<0.05). Real time reverse transcriptase polymerase chain reaction showed that morphine treatment (100 ng/ml) of hypoxic HUVECs resulted in a significant reduction in mRNA levels of VEGF121and VEGF165isoforms. Transfection of HUVECs with a human VEGF promoter–luciferase construct showed that hypoxia-induced transcriptional activation of VEGF was markedly inhibited by morphine sulfate (P<0.05). Phosphatidyl inositol-3 kinase and protein kinase C-mediated activation of the VEGF promoter was also inhibited by morphine. The opioid antagonist naloxone significantly reversed the inhibitory effects of morphine in endothelial cells suggesting the involvement of opioid receptors. Our results show that the inhibitory effects of morphine on hypoxia-induced VEGF expression in endothelial cells and cardiac myocytes can lead to a decrease in the autocrine and paracrine stimulation and hence limit neovascularization of the ischemic myocardium.

CONTRACTING SKELETAL MUSCLE CELLS EXPRESS AND RELEASE VEGF AND INDUCE VEGF MRNA IN ENDOTHELIAL CELLS

An important adaptation to endurance training is new capillary formation in skeletal muscle. Several growth factors may be involved in this angiogenic process, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). We sought to determine if contracting primary skeletal muscle cells express VEGF and/or bFGF mRNA and if muscle cells release VEGF. We also examined whether skeletal muscle cells produce compounds that are capable of enhancing synthesis of VEGF and bFGF in endothelial cells. Northern blot analysis of contracting primary skeletal muscle cells revealed existence of both VEGF and bFGF mRNA. Medium from contracting skeletal muscle cells was analysed for VEGF protein concentration and results indicated an increased secretion of VEGF protein into the medium from 0 hours to 37 hours of contraction. Human umbilical vein endothelial cells (HUVECs) were grown for 24 or 48 hours with (conditioned) or without medium transferred from contracting skeletal muscle cells. Northern blot analysis of endothelial cells grown for 48 hours with conditioned medium revealed a three-fold increase in VEGF mRNA expression compared to endothelial cells grown with non-conditioned medium (P < 0.05). No increase in VEGF mRNA could be detected after 24 hours of incubation (P > 0.05). Levels of bFGF mRNA in endothelial cells remained unaltered with time and with addition of conditioned medium (P > 0,05). The present data show that primary skeletal muscle cells express VEGF and bFGF mRNA and release VEGF protein. Furthermore it is demonstrated that contracting skeletal muscle cells produce not yet identified substance(s) that increase the expression of VEGF mRNA in endothelial cells. VEGF mRNA induction in the endothelial cells was time-dependent with maximal levels observed at 48 hours. These findings suggest that skeletal muscle cells participate in the regulation of angiogenesis both by releasing VEGF and by stimulating VEGF expression in endothelial cells.

Regulation of Vascular Endothelial Growth Factor Expression by cAMP in Rat Aortic Smooth Muscle Cells

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen which stimulates angiogenesis. VEGF is regulated by multiple factors such as hypoxia, phorbol esters, and growth factors. However, data concerning the expression of VEGF in the different vascular cell types and its regulation by cAMP are not available. In the present study, we have investigated the effect of adenylate cyclase activation on VEGF mRNA expression in rat vascular cells in primary culture. Basal VEGF expression is greater in smooth muscle cells than in endothelial cells and fibroblasts. A 4-h treatment with forskolin (10−5M) induced a 2-fold stimulation of VEGF mRNA expression in smooth muscle cells and fibroblasts, but, in contrast, did not affect VEGF expression in endothelial cells. In smooth muscle cells, a pharmacologically induced increase in intracellular cAMP levels using iloprost or isoprenaline led to a rise in VEGF mRNA expression comparable to that induced by forskolin. Adenosine, which increases cAMP levels in smooth muscle cells, also increases VEGF expression. Moreover, the 2.2-fold stimulation of VEGF expression by adenosine was enhanced following a cotreatment with cobalt chloride (a hypoxia miming agent). The observed additive effect (4.3-fold increase) suggests that these two factors, hypoxia and adenosine, regulate VEGF mRNA expression in smooth muscle cells by independent mechanisms.