Stimulated Vascular Smooth Muscle Cells Proliferation Research Articles

Osteogenic PTHs and vascular ossification—Is there a danger for osteoporotics?

Inflammation in vascular (mostly arterial) walls and heart valves triggered by the trans-endothelial influx of LDL particles and the action of subsequently modified (e.g., by oxidation) LDL particles can trigger true bone formation by valvar fibroblasts, by a subpopulation of re-differentiation-competent VSMCs (vascular smooth muscle cells) or by vascular pericytes. Vascular ossification can lead to heart failure and death. Elderly osteoporotic women who need osteogenic drugs to restore their lost skeletal bone are paradoxically prone to vascular ossification-the "calcification paradox." The recent introduction into the clinic of a potently osteogenic parathyroid hormone peptide, Lilly's rhPTH-(1-34)OH (Forteotrade mark), to reverse skeletal bone loss raises the question of whether this and other potently osteogenic PTHs still in clinical trial might also stimulate vascular ossification in such osteoporotic women. Indeed the VSMCs in human and rat atherosclerotic lesions hyperexpress PTHrP and the PTHR1 (or PTH1R) receptor as do maturing osteoblasts. And the evidence indicates that endogenous PTHrP with its NLS (nuclear/nucleolar localization sequence) does stimulate VSMC proliferation (a prime prerequisite for atheroma formation and ossification) via intranuclear targets that inactivate pRb, the inhibitory G1/S checkpoint regulator, by stimulating its hyperphosphorylation. But neither externally added full-length PTHrP nor the NLS-lacking PTHrP-(1-34)OH gets into the VSMC nucleus and instead they inhibit proliferation and calcification by only activating the cell's PTHR1 receptors. No PTH has an NLS and, as expected from the observations on the externally added PTHrPs, hPTH-(1-34)OH inhibits calcification by VSMCs and cannot stimulate vascular ossification in a diabetic mouse model. Encouraging though this may be for osteoporotics with their "calcification paradox," more work is needed to be sure that the skeletally osteogenic PTHs do not promote vascular ossification with its cardiovascular consequences.

Role of Parathyroid Hormone–Related Protein in the Regulation of Stretch-Induced Renal Vascular Smooth Muscle Cell Proliferation

In vivo, vascular smooth muscle cells (VSMC) are continuously exposed to mechanical cyclic stretch as a result of the pulsatile blood flow from the cardiac contractile cycle. Stretch is altered in pathologic conditions and contributes to vascular remodeling by modulating VSMC proliferation and death. Parathyroid hormone-related protein (PTHrP) is a locally produced poly-protein that regulates cell growth. It was shown previously that PTHrP inhibits VSMC proliferation through the auto/paracrine pathway by interacting with its receptor, the PTH1R, but stimulates VSMC proliferation through the intracrine pathway by translocating into the nucleus. In the current study, VSMC that were isolated from both resistance and compliance vessels were used to study the role of PTHrP in VSMC proliferation under experimental stretch. It is shown that PTHrP gene expression is upregulated by stretch and that PTHrP opposes the inhibitory effect induced by stretch on VSMC proliferation through the intracrine pathway. In addition, it is demonstrated that PTHrP expression is controlled at the post-transcriptional level by stretch. Taken together, these results strongly suggest that PTHrP plays a critical role in the modulation of VSMC proliferation in response to stretch. Thus, in conditions in which stretch is increased, such as in hypertension or in restenosis after angioplasty, PTHrP may contribute to vessel hyperplasia.

Endogenous VEGF-A is responsible for mitogenic effects of MCP-1 on vascular smooth muscle cells.

Vessel wall remodeling is a complex phenomenon in which the loss of differentiation of vascular smooth muscle cells (VSMCs) occurs. We investigated the role of rat macrophage chemoattractant protein (MCP)-1 on rat VSMC proliferation and migration to identify the mechanism(s) involved in this kind of activity. Exposure to very low concentrations (1-100 pg/ml) of rat MCP-1 induced a significant proliferation of cultured rat VSMCs assessed as cell duplication by the counting of total cells after exposure to test substances. MCP-1 stimulated VSMC proliferation and migration in a two-dimensional lateral sheet migration of adherent cells in culture. Endogenous vascular endothelial growth factor-A (VEGF-A) was responsible for the mitogenic activity of MCP-1, because neutralizing anti-VEGF-A antibody inhibited cell proliferation in response to MCP-1. On the contrary, neutralizing anti-fibroblast growth factor-2 and anti-platelet-derived growth factor-bb antibodies did not affect VSMC proliferation induced by MCP-1. RT-PCR and Western blot analyses showed an increased expression of either mRNA or VEGF-A protein after MCP-1 activation (10-100 pg/ml), whereas no fms-like tyrosine kinase (Flt)-1 receptor upregulation was observed. Because we have previously demonstrated that hypoxia (3% O2) can enhance VSMC proliferation induced by VEGF-A through Flt-1 receptor upregulation, the effects of hypoxia on the response of VSMCs to MCP-1 were investigated. Severe hypoxia (3% O2) potentiated the growth-promoting effect of MCP-1, which was able to significantly induce cell proliferation even at a concentration as low as 0.1 pg/ml. These findings demonstrate that low concentrations of rat MCP-1 can directly promote rat VSMC proliferation and migration through the autocrine production of VEGF-A.

Chlamydia pneumoniae stimulates proliferation of vascular smooth muscle cells through induction of endogenous heat shock protein 60.

Chlamydia pneumoniae infection has been linked with atherosclerosis. However, the mechanism responsible for the atherogenic effects of C pneumoniae remains unclear. Heat shock proteins (HSPs) have been found in atherosclerotic lesions. HSPs of HSP70 and HSP90 families are involved in the regulation of cell cycle progression and cell proliferation. We assessed the hypothesis that HSP60 is induced in vascular cells infected with C pneumoniae and stimulates cell proliferation. Rabbit vascular smooth muscle cells (VSMCs) and human umbilical vein endothelial cells (HUVECs) were infected with C pneumoniae. Western blot analysis demonstrated the induction of endogenous HSP60 expression in C pneumoniae-infected VSMCs. C pneumoniae infection significantly increased the number of VSMCs, and the mitogenic effect correlated with the expression level of endogenous HSP60. In contrast to VSMCs, C pneumoniae infection had no effect on the expression level of HSP60 and did not stimulate cell proliferation in HUVECs. Exogenous addition of recombinant chlamydial HSP60 had no mitogenic effect on VSMCs and HUVECs. However, overexpression of HSP60 within VSMCs by infection with adenovirus encoding human HSP60 resulted in a significant increase in cell numbers compared with uninfected VSMCs. These results suggest that overexpression of endogenous HSP60 may be a central intracellular event responsible for the mitogenic effects induced by C pneumoniae infection. In addition to C pneumoniae, other infectious agents and atherogenic risk factors may also stimulate VSMC proliferation and contribute to the lesion formation through the induction of HSP60.

Influence of insulin and of insulin resistance on platelet and vascular smooth muscle cell function

In this short review, we present the main results obtained in our laboratory in the last 15 years concerning the influence exerted by insulin on platelets and human vascular smooth muscle cells (VSMCs). In particular, we discuss: (i) the insulin ability to rapidly activate a constitutive nitric oxide synthase (NOS) in both cell types, with a consequent increase of the two nucleotides guanosine-3′,5′-cyclic monophosphate (cGMP) and adenosine-3′,5′-cyclic monophosphate (cAMP), well-known mediators of antiaggregation and vasodilation; (ii) the interplay of insulin with substances able to activate adenylate cyclase in both cell types; (iii) the impairment of the antiaggregating insulin effects in insulin-resistant subjects; (iv) the insulin-induced increase on endothelin in the VSMCs; (v) the insulin ability to slightly stimulate VSMC proliferation.

Activation of mitogenic and antimitogenic pathways in cyclically stretched arterial smooth muscle.

Biophysical forces regulate vascular smooth muscle cell (VSMC) physiology and evoke vascular remodeling. Two VSMC autocrine molecules, insulin-like growth factor I (IGF-I) and nitric oxide (NO), are implicated in remodeling attributable to VSMC hyperplasia. We investigated the role of in vitro cyclic stretch on rat VSMC IGF-I, NO, and cellular growth. Cyclic stretch (1 Hz at 120% resting length for 48 h) stimulated VSMC proliferation 2.5-fold vs. unstretched cells and was accompanied by a 1.8-fold increase in VSMC IGF-I secretion. Despite activation of this proliferative pathway, cyclic stretch induced inducible (i) nitric oxide synthase (NOS) expression and a twofold increase in NO secretion, a molecule with documented antiproliferative effects. Cytokine treatment enhanced iNOS expression and NO secretion while inhibiting vascular growth by approximately 50% in static cells. Cytokine treatment of stretched VSMC enhanced NO secretion 2.5-fold while inhibiting growth by approximately 80%. Exogenous IGF-I increased NOS activity 1.5-fold and NO secretion 8.5-fold in static cells. In turn, iNOS inhibition increased IGF-I secretion 1.6-fold and enhanced VSMC growth 1.6-fold in stretched cells. An NO donor (sodium nitroprusside) similarly inhibited VSMC proliferation in static (24%) and stretched (50%) VSMC while also inhibiting IGF-I secretion from stretched cells by approximately 35%. Thus cyclic stretch stimulates mitogenic (IGF-I) and antimitogenic (NO) pathways in VSMC. These two molecules regulate each other's secretory rates, providing tight regulation of VSMC proliferation. These data may have profound implications in understanding vascular growth alterations in vascular injury and hypertension.

Chlamydia pneumoniae and chlamydial heat shock protein 60 stimulate proliferation of human vascular smooth muscle cells via toll-like receptor 4 and p44/p42 mitogen-activated protein kinase activation.

An early component of atherogenesis is abnormal vascular smooth muscle cell (VSMC) proliferation. The presence of Chlamydia pneumoniae in many atherosclerotic lesions raises the possibility that this organism plays a causal role in atherogenesis. In this study, C pneumoniae elementary bodies (EBs) rapidly activated p44/p42 mitogen-activated protein kinases (MAPKs) and stimulated proliferation of VSMCs in vitro. Exposure of VSMCs derived from human saphenous vein to C pneumoniae EBs (3x10(7) inclusion forming units/mL) enhanced bromodeoxyuridine (BrdU) incorporation 12+/-3-fold. UV- and heat-inactivated C pneumoniae EBs also stimulated VSMC proliferation, indicating a role of direct stimulation by chlamydial antigens. However, the mitogenic activity of C pneumoniae was heat-labile, thus excluding a role of lipopolysaccharide. Chlamydial hsp60 (25 microg/mL) replicated the effect of C pneumoniae, stimulating BrdU incorporation 7+/-3-fold. Exposure to C pneumoniae or chlamydial hsp60 rapidly activated p44/p42 MAPK, within 5 to 10 minutes of exposure. In addition, PD98059 and U0126, which are two distinct inhibitors of upstream MAPK kinase 1/2 (MEK1/2), abolished the mitogenic effect of C pneumoniae and chlamydial hsp60. Toll-like receptors (TLRs) act as sensors for microbial antigens and can signal via the p44/p42 MAPK pathway. Human VSMCs were shown to express TLR4 mRNA and protein, and a TLR4 antagonist abolished chlamydial hsp60-induced VSMC proliferation and attenuated C pneumoniae-induced MAPK activation and VSMC proliferation. Together these results indicate that C pneumoniae and chlamydial hsp60 are potent inducers of human VSMC proliferation and that these effects are mediated, at least in part, by rapid TLR4-mediated activation of p44/p42 MAPK.

Differential ANG II-induced growth activation pathways in mesenteric artery smooth muscle cells from SHR.

Angiotensin II-induced growth signaling mechanisms were investigated in vascular smooth muscle cells (VSMCs) from mesenteric arteries of spontaneously hypertensive (SHR) and Wistar-Kyoto rats (WKY). In WKY, angiotensin II significantly increased protein synthesis ([(3)H]leucine incorporation) but not DNA synthesis ([(3)H]thymidine incorporation). In SHR, angiotensin II increased protein and DNA synthesis. VSMCs from both strains expressed angiotensin type 1 (AT(1)) and type 2 (AT(2)) receptors. Losartan (an AT(1) receptor antagonist) but not PD-123319 (an AT(2) receptor antagonist) attenuated angiotensin II-stimulated protein synthesis in WKY VSMCs. In SHR, losartan and PD-123319 partially inhibited angiotensin II-induced VSMC proliferation. The mitogen-activated protein kinase or extracellular signal-regulated protein kinase (ERK) kinase inhibitor PD-98059 blocked VSMC growth responses to angiotensin II in both strains. Angiotensin II increased ERK1/2 activation more in SHR than WKY, an effect inhibited by losartan but not PD-123319. LY-294002 [a phosphatidylinositol-3 (PI3) kinase inhibitor] blocked angiotensin II-stimulated ERK1/2 activation in SHR but not in WKY, whereas bisindolylmaleimide [a protein kinase C (PKC) inhibitor] was ineffective. In conclusion, angiotensin II stimulates VSMC proliferation via AT(1) and AT(2) receptors in SHR. In WKY, angiotensin II induces VSMC hypertrophy via AT(1) receptors. ERK1/2-dependent pathways regulated by intracellular Ca(2+) but not PKC mediate these effects. In SHR VSMCs, PI3 kinase plays a role in augmented angiotensin II-induced ERK1/2 phosphorylation. These angiotensin II-mediated signaling events could contribute to vascular remodeling in SHR.

ANG II potentiates mitogenic effect of norepinephrine in vascular muscle cells: role of FGF-2.

We examined the possible cooperation between norepinephrine (NE) and ANG II on proliferation of cultured vascular smooth muscle cells (VSMCs) and the involved cellular mechanisms. Nanomolar NE concentrations stimulated VSMC proliferation through a prazosin-sensitive effect. The pretreatment of cells with 100 nM ANG II for 24 h significantly potentiated the NE-induced VSMC proliferation; this potentiating effect of ANG II was blocked by losartan but was unaffected by the AT(2) receptor antagonist PD-123177. ANG II pretreatment also potentiated the increase in inositol phosphate turnover and upregulated the cell expression of fibroblast growth factor (FGF-2) induced by NE. Anti-FGF-2 neutralizing antibodies prevented the potentiating effect of ANG II on NE-induced cell growth. Both ANG II and NE stimulated extracellular signal-related kinase (ERK1) activation, but an ANG II potentiation of the effect of NE on ERK1 activity was not detectable. Moreover, ANG II significantly increased protein synthesis but did not potentiate the hypertrophic effect of NE. These findings demonstrate that ANG II and NE cooperate in promoting VSMC growth and that FGF-2 upregulation is involved in this effect.

Endothelin-1 is involved in the growth promotion of vascular smooth muscle cells by hyaluronic acid

Proliferation of vascular smooth muscle cells (VSMC) is crucial to the progression of arteriosclerosis. In this study, we examined the role that interactions among endothelin-1 (ET-1), CD44, and hyaluronic acid (HA) play in VSMC proliferation. Co-localization of ET-1, CD44 and HA positive areas, as well as proliferating cell nuclear antigen positive nuclei, were observed in mouse neointima induced by endothelial injury. As found in intimal VSMC, cultured mouse VSMC secreted ET-1. The endothelin-converting enzyme (ECE) inhibitor, phosphoramidon, and endothelin type-A (ETA) receptor antagonist BQ-123 reduced expression of CD44 in VSMC. ET-1 reversed the reduction of CD44 expression by phosphoramidon. Because CD44 is a receptor for HA, we investigated the effects of phosphoramidon, BQ-123 or ET-1 on the mitogenic activity of HA in VSMC. Among the different molecular weights of this polysaccharide, oligosaccharides of HA (oHA) stimulated VSMC proliferation most effectively. Phosphoramidon and BQ-123 inhibited this oHA-induced DNA synthesis in VSMC. ET-1 reversed the suppression of oHA-induced proliferation by phosphoramidon. In conclusion, endogenously secreted ET-1 enhances oHA-stimulated VSMC growth via the ETA receptor in an autocrine manner. Thus it is suggested that the CD44-inducing activity of ET-1 is responsible for its stimulating effect on oHA-dependent growth of VSMC. These findings support the hypothesis that the interactions among ET-1, CD44 and HA promote the progression of arteriosclerosis.

Chlamydia pneumoniae activates nuclear factor kappaB and activator protein 1 in human vascular smooth muscle and induces cellular proliferation.

Observational data strongly suggest an association between Chlamydia pneumoniae and atherosclerotic cardiovascular disease. However, few studies have mechanistically linked C. pneumoniae to vascular remodeling. The purpose of the present study was to examine the mechanistic relationship between C. pneumoniae and human vascular smooth muscle cell (VSMC) physiology. We sought to determine the influence of human VSMC infection by C. pneumoniae on (1) VSMC proliferation and (2) activation of the proinflammatory and proliferative transcription factors nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1). C. pneumoniae was grown and isolated from Hep 2 cells. Human aortic VSMCs were inoculated with C. pneumoniae in the presence and absence of the azalide antibiotic azithromycin. Cell proliferation was assayed by direct cell counting 48 h following infection. Two hours following infection, nuclear extracts were isolated, and activation of both NF-kappaB and AP-1 was assessed by electrophoretic mobility shift assay. Compared with control, C. pneumoniae infection stimulated VSMC proliferation (P < 0.05) and induced both NF-kappaB and AP-1 DNA binding activity. These effects were eliminated by concurrent treatment with azithromycin. VSMC infection with C. pneumoniae activates proliferative intracellular signals and stimulates cell growth. These data implicate C. pneumoniae as a pathogenic mediator and a potential therapeutic target in the prevention of atherosclerotic disease.

Age-dependent increase in c-fos activity and cyclin A expression in vascular smooth muscle cells. A potential link between aging, smooth muscle cell proliferation and atherosclerosis.

Aging can be defined as a progressive deterioration of biological functions after the organism has attained its maximal reproductive competence, which is usually associated with a decrease in proliferative ability in most cell types. However, in certain pathological situations such as atherosclerosis and restenosis, aging has been shown to be associated with a higher level of vascular smooth muscle cell (VSMC) proliferation and neointimal lesion formation after angioplasty. In the present study, we investigated potential mechanisms involved in the age-dependent increase in VSMC proliferation. Primary cultures of VSMCs were isolated from young (6-8-month-old) and old (4-5-year-old) New Zealand rabbits. Results from cell counting assays and FACS analysis were consistent with a shortening of the cell cycle in old VSMCs. Western blot analysis in serum stimulated cells showed a significant increase in the level of cyclin A and cyclin-dependent kinase 2 proteins in the old vs. young VSMCs. In marked contrast, expression of cyclin E in VSMCs was not influenced by aging. Transient transfection assays showed an age-dependent increase in transcription from the human cyclin A promoter. Parallel studies demonstrated that the expression of the AP1 transcription factor c-fos, which interacts with the cyclin A promoter and stimulates VSMC proliferation, was also increased in old VSMCs. Consistent with this notion, electrophoretic mobility shift assays demonstrated an increase in AP1 DNA-binding activity in old VSMCs. These studies suggest that age-associated increase in c-fos activity contributes to augmented cyclin A expression and VSMC proliferation in old animals. These mechanisms might contribute to the higher prevalence and severity of atherosclerosis in the elderly.

Inhibition of estrogen receptor function promotes porcine coronary artery smooth muscle cell proliferation

Estradiol-17β (E 2) can inhibit vascular smooth muscle cell (VSMC) proliferation probably through its ability to activate its nuclear estrogen receptors (ER). Activation or inhibition of the ER by cognate permissive or non-permissive ligands, respectively, would indicate whether ER action is critical for this vascular protective effect. We investigated a previously characterized population of cultured porcine coronary artery SMCs for ER expression and for the response of these cells to estrogens and antiestrogens. Reverse transcription-polymerase chain reaction and Western blot analyses demonstrated ER mRNA and protein, respectively, in these cells. While the culture conditions required may have prevented the demonstration of physiological effects of E 2, the antiestrogens, ICI 182,780 and 4-hydroxytamoxifen, stimulated VSMC proliferation. The data suggest that, by interrupting ER function, antiestrogens significantly increased the VSMC mitotic rate. This model may be used to identify ER-regulated genes that function to control the growth of these coronary artery SMCs.

Thrombospondin-1 induces activation of focal adhesion kinase in vascular smooth muscle cells

Purpose: Vascular smooth muscle cell (VSMC) proliferation and migration are important events in the development of intimal hyperplasia. Thrombospondin-1 (TSP-1), an extracellular matrix protein present in intimal hyperplastic lesions, has been shown to stimulate VSMC proliferation and migration. We hypothesized that the focal adhesion plaque, specifically the focal adhesion kinase (FAK) protein, may be important in the signal transduction pathway for TSP-1-induced VSMC migration. Methods: Growth-arrested bovine aortic VSMCs were treated with TSP-1 (20 μg/mL) for set intervals (15, 30, and 120 minutes) and compared with VSMCs grown in serum-free medium (negative control) or in the presence of a known mitogen and chemotactic factor, platelet-derived growth factor (10 ng/mL; positive control). Crude cell lysates and anti-FAK immunoprecipitates were assayed for phosphotyrosine activity by means of antiphosphotyrosine immunoblotting. The blots were quantified by means of densitometric analysis. Results: TSP-1 increased tyrosine phosphorylation of three protein bands of molecular weights 68, 125 (consistent with FAK), and 180 kDa. Immunoprecipitation with FAK antibody, followed by antiphosphotyrosine immunoblotting, indicated that there was an increase in tyrosine phosphorylation of FAK at 15, 30, and 120 minutes in the TSP-1-treated groups ( P < .05). Conclusion: Tyrosine phosphorylation of FAK is induced by TSP-1 stimulated VSMCs. This suggests an outside-inside signaling pathway by which TSP-1 stimulates VSMC migration. (J Vasc Surg 1999;29:1031-6.)

CYCLOSPORINE A STIMULATION OF VASCULAR SMOOTH MUSCLE CELL PROLIFERATION IS ASSOCIATED WITH INCREASED EXPRESSION OF 14-3-3γ PROTEIN.

235 Cyclosporine A (CyA) is a potent immunosuppressant that has significantly improved graft survival in organ and bone marrow transplant recipients. However, CyA may accelerate vascular disease by stimulating vascular smooth muscle cell (VSMC) proliferation. While high CyA concentrations inhibit proliferation of many cells, low in vitro concentrations which mimic clinical trough blood levels stimulate cellular proliferation. The mechanism by which CyA stimulates cellular proliferation is unknown. The kinase chaperone protein 14-3-3γ is highly expressed in VSMCs and helps regulate cell cycle progression and cellular proliferation. We hypothesized that CyA increases VSMC proliferation by a mechanism that requires increased 14-3-3γ expression. VSMCs were isolated from rat aorta. Cell purity was confirmed by staining of α smooth muscle actin. Cellular proliferation was determined by the colorimetric assay MTT using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). 14-3-3γ expression was measured in VSMCs by immunoblotting. (*p<0.05 T test) There was a concentration-dependent proliferation response to 3 day exposure to CyA (O.D. @ 570 nm normalized to control): CyA 10−8M [1.22]*, CyA 10−7 M [1.15], CyA 10−5 M [1.01], PHA 1:200 [1.06]. 14-3-3γ expression was increased by CyA in a concentration-dependent fashion (Density of immunoblot normalized to control): CyA 10−9 [2], CyA 10−8 M [5], CyA 10−7 M [4], CyA 10−5 M [3.7] vs control. CyA also stimulated VSMC proliferation and 14-3-3γ expression in a time-dependent fashion. Proliferation was significantly increased after 3 days of exposure to CyA 10−7 M [1.29] vs control at 3d*, and day 3 vs day 1 CyA 10−7 M [1.16]. 14-3-3γ expression was increased early after exposure to CyA 10−7 M 8hr [3.5] (expression at 6,16,24 hr n.s. vs control) but not late (14-3-3γ expression at 3d [1.05] PHA at 3d [1.00]). In summary, CyA 10−7 M significantly stimulated VSMC proliferation in a time-dependent manner. CyA stimulated 14-3-3γ expression in a concentration dependent manner. Stimulation of 14-3-3γ expression may be a significant signal transduction mechanism by which CyA controls VSMC proliferation.

The additive effects of glucose and insulin on the proliferation of infragenicular vascular smooth muscle cells

Purpose: Peripheral vascular disease involving the infragenicular arterial tree is common in patients with diabetes mellitus (DM). Accelerated proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development of atherosclerosis. Insulin and glucose stimulate VSMC proliferation and are elevated in patients with non–insulin-dependent DM. We have previously described the mitogenic effect of insulin on VSMCs in vitro; the effects of insulin and glucose separately and in combination on the proliferation of VSMCs grown in serum-free media were studied. Methods: Human infragenicular VSMCs isolated from diabetic patients with end-stage peripheral vascular disease undergoing below-knee amputation were used. Cells from passages 3 to 5 were grown in serum-free media with varying glucose (0.05%, 0.1%, 0.2%, 0.4%, 0.6%, and 0.8%) and insulin (no added insulin, 100 ng/mL, and 1000 ng/mL) concentrations for 6 days. Results: Insulin stimulated VSMC growth at glucose concentrations more than 0.2% (0.4% glucose with no added insulin resulted in 13,073 ± 336 cells/mL, 0.4% glucose with 100 ng/mL insulin resulted in 16,536 ± 1175 cells/mL, 0.4% glucose with 1000 ng/mL insulin resulted in 17,500 ± 808 cells/mL, 0.6% glucose with no added insulin resulted in 14,167 ± 1062 cells/mL, 0.6% glucose with 100 ng/mL insulin resulted in 18,984 ± 1265 cells/mL, 0.6% glucose with 1000 ng/mL insulin resulted in 20,450 ± 1523 cells/mL, 0.8% glucose with no added insulin resulted in 15,853 ± 1650 cells/mL, 0.8% glucose with 1000 ng/mL insulin resulted in 26,302 ± 1919 cells/mL; P < .05 compared with glucose with no added insulin). Glucose stimulated VSMC proliferation up to a concentration of 0.2% (42% and 117% higher growth at 0.1% and 0.2% glucose, respectively, compared with the baseline, P < .05), regardless of the insulin concentration in the media. The greatest growth (26,302 ± 1919 cells/mL) occurred in the group with the highest concentration of both insulin (1000 ng/mL) and glucose (0.8% glucose; P < .05). Conclusion: Both insulin and glucose stimulate the growth of diabetic infragenicular VSMCs. The mitogenic effects of insulin and glucose are additive and may contribute to the development of atherosclerosis in patients with DM. (J Vasc Surg 1998;28:1033-9.)