Rabbit Arterial Smooth Muscle Cells Research Articles

Deposition of organic−inorganic hybrid coatings over 316L surgical stainless steel and evaluation on vascular cells

Surface coating of metallic materials using the sol-gel technique is a suitable approach to obtain hybrid materials with improved properties for biomedical applications. In this study, an AISI 316L stainless steel surface was coated with ormosils prepared from tetraethylsiloxane and 3-glycidoxypropyltrimethoxysilane or polydimethylsiloxane. The characterization of structural and surface properties was performed by several techniques. Surface microstructure, morphology, and energy are dependent on organosilane type and content. Chemical stability of coatings was investigated by static immersion tests in phosphate buffer solution at 37 °C, and silicon leaching after 21 days was found to be in the range of ∼200−300 μg L−1. Mechanical adhesion was found to be within 1.0 and 3.7 N cm−1. The interaction of the samples and materials in the cardiovascular environment was investigated through cellular behavior. Biological assays were performed with slides to avoid any cytotoxic effects on human endothelial cells (HUVEC) and rabbit arterial smooth muscle cells (RASM). No significant alterations were observed after 24 h in the viability of RASM and HUVEC cells exposed to different coatings. No increase of HUVEC or RASM migration was observed after 24 h as evaluated by transwell migration assay. The hybrid materials showed suitable properties for potential application as biomaterials in cardiovascular environment as well as for incorporation of bioactive species with the aim to prepare drug-eluting stents.

Abstract 507: G Proteins are Required for Lipoxygenase EDHF Activity of Rabbit Arterial Smooth Muscle Cells

Arachidonic acid 15-lipoxygenase (15-LO) metabolites function as endothelium-derived hyperpolarizing factors in rabbit and human arteries. In rabbit arteries, LO metabolites mediate nitric-oxide and prostaglandin-independent relaxations to acetylcholine and AA. Previously, we characterized 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA) as a major vasoactive 15-LO metabolite in rabbit arteries. 11,12,15-THETA requires a specific structure for vascular activity. 11(R),12(S),15(S)-THETA causes concentration-related relaxation whereas 11(R),12(R),15(S)-THETA is without activity. The specific structure requirement suggests a role for a receptor. Therefore, we examined the role of G proteins in 11(R),12(S),15(S)-THETA vascular activity. Western immunoblot verified protein expression of Gαs, Gαi and a Gαo in rabbit endothelial and smooth muscle cells. 11(R),12(S),15(S)-THETA increased GTPγ35S binding to rabbit arterial membranes 280±25% while 11(R),12(S),15(S)-THETA was without effect. In cell-attached patches of rabbit smooth muscle, 11(R),12(S),15(S)-THETA (100 nM) increased mean open time of apamin-sensitive, calcium-activated, small conductance potassium (SK) channels from 0.0001±0.0001 to 0.0015±0.0006. In inside-out patches, 11(R),12(S),15(S)-THETA did not increase channel opening (0.0001±0.0001) unless GTP was present (0.0051±0.0023). In the presence of GTP, an antibody against Gαs and a Gαs inhibitory peptide inhibited 11(R),12(S),15(S)-THETA SK channel activation (0.0007±0.0005, 0.0013±0.0012, respectively) whereas an antibody against Gαi was without effect (0.0042±0.0018). A cell-permeant, penetratin-linked Gαs inhibitory peptide also inhibited 11(R),12(S),15(S)-THETA SK channel activation in cell-attached patches (0.0005±0.0002) and blocked 11(R),12(S),15(S)-THETA relaxations in rabbit aorta (max relaxations = 74±6%, 23±7% for control and permeant peptide, respectively). These studies indicate that 11,12,15-THETA-induced SK channel activation and vascular relaxation are mediated by a Gs-coupled mechanism and that 11,12,15-THETA acts via a stereo-specific G protein coupled receptor/binding site.

In vitro evaluation of the safe margin, antithrombotic and antiproliferative actions for the treatment of restenosis: Nitric oxide donor and polymers

Drug-eluting stents (DES) were developed to combat the problem of in-stent restenosis, and evaluating the biological activity from DES systems is critical for its safety and efficacy. To test the cytotoxicity of nitric oxide (NO) donor-containing polymers for their potential use in DES applications, S-nitrosoglutathione (GSNO) or in combination with poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) in an aqueous polymeric solution (PVA/PVP/GSNO) was investigated using Balb/c 3T3 and Rabbit arterial smooth muscle (RASM) cells. The sensitivity of 3T3 cells to the cytotoxicity effects induced by GSNO was higher than that of RASM cells, while RASM cells were more susceptible to alterations in membrane permeability. Cell growth assays showed that GSNO and PVA/PVP/GSNO induced antiproliferative effects in RASM cells. Moreover, the presence of polymers can reduce the cytotoxicity and enhance the antiproliferative effects of GSNO. Dose-dependent inhibition of platelet aggregation was similar for both PVA/PVP/GSNO (EC50 of 3.4 ± 2.3 µM) and GSNO (EC50 of 2.8 ± 1.1 µM) solutions. Platelet adhesion assays showed that the inhibition caused by GSNO (EC50 of 5.0 mM) was dependent on the presence of plasma. These results demonstrate that the methodology adopted here is suitable to establish safety margins and evaluate the antithrombotic potential and antiproliferative effects of NO-eluting biomaterials and polymeric solutions for the new cardiovascular devices, and also to emphasize the importance of using more specific cell lines in these evaluations.

Inhibition of human and rabbit arterial smooth muscle cell migration mediated by the kinin B1 receptor: role of receptor density and released mediators

Bradykinin (BK)-related peptides are suspected to negatively influence diverse functions in vascular smooth muscle cells (SMCs), notably via stimulation of the inducible B1 receptor (B1R), and have been shown to inhibit the migration of rat SMCs. The present study had several objectives: (i) to test whether B1R mediates the inhibition of migration of arterial SMCs from additional species (the human and the rabbit); (ii) whether B1R density influences this action and whether autocrine NO or prostanoid release modulate it; and (iii) the possible signaling interaction between the B1R and phosphatidylinositol-3 kinase (PI-3K) has been addressed. The peptidase resistant B1R agonist Sar-[D-Phe8]des-Arg9-BK (10 nmol/L - 1 micromol/L) was an inhibitor of migration in human or rabbit arterial SMCs in a wound closure assay, more effectively if the medium composition allowed a high B1R expression (20% fetal bovine serum (FBS) + interleukin-1beta (IL-1beta) in human SMCs, 10% FBS in rabbit cells). The effect of the B1R agonist on motility was abrogated by a B1R antagonist, B-9858, but not by the B2R antagonist Hoe 140; a peptidase-resistant B2R agonist, [Phe8Psi(CH2-NH)-Arg9]BK, had a marginal or no effect on migration. Sar-[D-Phe8]des-Arg9-BK (1 micromol/L) did not significantly influence SMC proliferation. The B1R-mediated inhibition of SMC migration was not affected by pharmacological inhibition of the nitric oxide synthases or cyclooxygenases-1 or -2, but was correlated to an inhibition of PI-3K in both types of SMCs. The inhibition of SMC migration mediated by the kinin B1R is likely independent from NO or prostanoid release, applicable to several species, and correlated to receptor density and the inhibition of PI-3K.

Discovery of a Dual-Function Peptide That Combines Aminopeptidase N Inhibition and Kinin B1Receptor Antagonism

Previous analyses support that aminopeptidase N is a major inactivation pathway for high-affinity peptide ligands of the human and rabbit forms of the kinin B(1) receptor (agonists or antagonists). In this study, we found that the high-affinity antagonist B-9958 (Lys-Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-BK; des-Arg(9)-BK, des-arginine(9)-bradykinin) is an aminopeptidase N substrate based on its capacity to compete for the hydrolysis of the chromogenic substrate L-Ala-p-nitroanilide by membranes isolated from human or rabbit arterial smooth muscle cells, its inactivation in the presence of these membranes (radioreceptor assay) and on its intense potentiation by the aminopeptidase N inhibitor amastatin in the rabbit aorta contractility assay (gain of 0.84 units in the pA(2) scale). Analogs of B-9958 in which the N-terminal Lys residue was substituted by D-Lys or D-Arg (B-10352 and B-10356, respectively) showed reduced affinity at the human or rabbit B(1) receptors (1.2-2.8-fold), as estimated by the displacement of [(3)H]Lys-des-Arg(9)-BK binding, but were more potent antagonists of des-Arg(9)-BK-induced contraction of the rabbit aorta than B-9958 in the absence of amastatin; they were not potentiated by the latter inhibitor. Unexpectedly, B-10356 inhibited L-Ala- p-nitroanilide hydrolysis without being inactivated, suggesting that it is an aminopeptidase N inhibitor. This was verified because B-10356 (but not B-10352) potentiated peptides unrelated to kinins but susceptible to aminopeptidase N inactivation (angiotensin III, thrombin receptor hexapeptide agonist). B-10356 inhibits dual molecular targets (aminopeptidase N enzyme K(i), 0.9-2.2 microM; kinin B(1) receptor binding K(i), 0.5-1.5 nM), and this may be an advantage for specific therapeutic applications (e.g., inhibition of angiogenesis).

Shortening velocity and myosin heavy- and light-chain isoform mRNA in rabbit arterial smooth muscle cells.

In smooth muscle cells (SMCs) isolated from rabbit carotid, femoral, and saphenous arteries, relative myosin isoform mRNA levels were measured in RT-PCR to test for correlations between myosin isoform expression and unloaded shortening velocity. Unloaded shortening velocity and percent smooth muscle myosin heavy chain 2 (SM2) and myosin light chain 17b (MLC(17b)) mRNA levels were not significantly different in single SMCs isolated from the luminal and adluminal regions of the carotid media. Saphenous artery SMCs shortened significantly faster (P < 0.05) than femoral SMCs and had more SM2 mRNA (P < 0.05) than carotid SMCs and less MLC(17b) mRNA (P < 0.001) and higher tissue levels of SMB mRNA (P < 0.05) than carotid and femoral SMCs. No correlations were found between percent SM2 and percent MLC(17b) mRNA levels and unloaded shortening velocity in SMCs from these arteries. We have previously shown that myosin heavy chain (MHC) SM1/SM2 and SMA/SMB and MLC(17a)/MLC(17b) isoform mRNA levels correlate with protein expression for these isoforms in rabbit smooth muscle tissues. Thus we interpret these results to suggest that 1) SMC myosin isoform expression and unloaded shortening velocity do not vary with distance from the lumen of the carotid artery but do vary in arteries located longitudinally within the arterial tree, 2) MHC SM1/SM2 and/or MLC(17a)/MLC(17b) isoform expression does not correlate with unloaded shortening velocity, and 3) intracellular expression of the MHC SM1/SM2 and MLC(17a)/MLC(17b) isoforms is not coregulated.

Mildly oxidized LDL induces activation of platelet-derived growth factor beta-receptor pathway.

Mildly oxidized LDL (moxLDL) is thought to play a role in atherogenesis. MoxLDL induces derivatization of cell proteins and triggers a variety of intracellular signaling. We aimed to investigate whether moxLDL-induced protein derivatization may influence the activity of platelet-derived growth factor receptor beta (PDGFRbeta), a tyrosine kinase receptor of major importance in vascular biology and atherogenesis. In cultured rabbit arterial smooth muscle cells, moxLDL induces activation of the PDGFRbeta signaling pathway, as shown by PDGFRbeta tyrosine phosphorylation on Western blot and coimmunoprecipitation of SH2-containing proteins. The cellular events involved in the moxLDL-induced PDGFRbeta activation can be summarized as follows. Oxidized lipids from moxLDL trigger two phases of PDGFRbeta activation involving two separate mechanisms, as shown by experiments on cultured cells (in situ) and on immunopurified PDGFRbeta (in vitro): (1) the first phase may be mediated by 4-hydroxynonenal, which induces PDGFRbeta adduct formation and subsequent PDGFRbeta activation (antioxidant-insensitive step); (2) the second phase involves ceramide-mediated generation of H(2)O(2) (these steps being inhibited by tosylphenylalanylchloromethylketone, an inhibitor of ceramide formation, and by antioxidant BHT, exogenous catalase, or overexpressed human catalase). Because 4-hydroxynonenal-PDGFRbeta adducts are also detected in atherosclerotic aortas, it is suggested that this novel mechanism of moxLDL-induced PDGFRbeta activation may occur during atherogenesis. MoxLDL acts as a local autoparacrine mediator in the vascular wall, and PDGFRbeta acts as a sensor for both oxidized lipids and oxidative stress. This constitutes a novel mechanism of PDGFRbeta activation in atherosclerotic areas.

Differential regulation of Ca(2+)-activated Cl(-) currents in rabbit arterial and portal vein smooth muscle cells by Ca(2+)-calmodulin-dependent kinase.

1. Ca(2+)-activated chloride currents (I(Cl(Ca))) were recorded from smooth muscle cells isolated from rabbit pulmonary (PA) and coronary artery (CA) as well as rabbit portal vein (PV). The characteristics and regulation by Ca(2+)-calmodulin-dependent kinase II (CaMKII) were compared between the three cell types. 2. In PA and CA myocytes dialysed and superfused with K+ -free media, pipette solutions containing fixed levels of free Ca(2+) in the range of 250 nM to 1 microM evoked well sustained, outwardly rectifying I(Cl(Ca)) currents in about 90 % of cells. The CaMKII inhibitor KN-93 (5 microM) increased the amplitude of I(Cl(Ca)) in PA and CA myocytes. However, the threshold intracellular Ca(2+) concentration for detecting this effect was different in the two arterial cell types. KN-93 also enhanced the rate of activation of the time-dependent current during depolarising steps, slowed the kinetics of the tail current following repolarisation, and induced a negative shift of the steady-state activation curve. 3. In PA myocytes, the effects of KN-93 were not mirrored by its inactive analogue KN-92 but were reproduced by the inclusion of autocamtide-2-related CaMKII inhibitory peptide (ARIP) in the pipette solution. Cell dialysis with constitutively active CaMKII (30 nM) significantly reduced I(Cl(Ca)) evoked by 500 nM Ca(2+). 4. In PV myocytes, I(Cl(Ca)) was evoked by pipette solutions containing up to 1 microM free Ca(2+) in less than 40 % of cells. Application of KN-93 to cells where I(Cl(Ca)) was sustained produced a small inhibition (approximately 25%) of the current in 70 % of the cells. 5. The present study shows that regulation of Ca(2+)-dependent Cl(-) channels by CaMKII differs between arterial and portal vein myocytes.

Chylomicron-remnant-induced foam cell formation and cytotoxicity: a possible mechanism of cell death in atherosclerosis

The effects of chylomicron remnants on cytoplasmic lipid loading and cell viability were assessed in cultures of human monocyte-derived macrophages and rabbit arterial smooth muscle cells. At a cholesterol concentration of 150 microg/ml, chylomicron remnants induced substantial cytoplasmic lipid loading of macrophages, but not of smooth muscle cells, within 6 h of exposure. Chylomicron remnants were found to be cytotoxic to macrophages and smooth muscle cells, although the latter were generally more resistant. Chylomicron remnants contained no detectable oxysterols (>1 ng) and contained less non-esterified ('free') fatty acids than non-lipolysed nascent chylomicrons. Chylomicron-remnant-induced cytotoxicity appeared to be time- and dose-dependent. Macrophage and smooth muscle cell viability were inversely related to the production of superoxide free radicals and were significantly improved in the combined presence of superoxide dismutase and catalase. Collectively, our data suggest that, in macrophages, cell viability is compromised as a consequence of superoxide free radical production following uptake of chylomicron remnants. We would suggest that, in arterial smooth muscle cells, chylomicron-remnant-induced cell death also occurs as a consequence of superoxide free radical production. Our observations in the present study suggest that macrophage foam cells in atherosclerotic plaques might be derived from the cellular uptake of chylomicron remnants. Furthermore, arterial accumulation of chylomicron remnants might contribute to plaque destabilization as a consequence of cell death following superoxide free radical production by macrophages and smooth muscle cells.

P-344 - Effects of KRN2391 and KRN4884 on membrane currents in rabbit arterial smooth muscle cells

P-344 - Effects of KRN2391 and KRN4884 on membrane currents in rabbit arterial smooth muscle cells

Expression of smooth muscle myosin heavy chains and unloaded shortening in single smooth muscle cells.

The functional significance of the variable expression of the smooth muscle myosin heavy chain (SM-MHC) tail isoforms, SM1 and SM2, was examined at the mRNA level (which correlates with the protein level) in individual permeabilized rabbit arterial smooth muscle cells (SMCs). The length of untethered single permeabilized SMCs was monitored during unloaded shortening in response to increased Ca2+ (pCa 6.0), histamine (1 microM), and phenylephrine (1 microM). Subsequent to contraction, the relative expression of SM1 and SM2 mRNAs from the same individual SMCs was determined by reverse transcription-polymerase chain reaction amplification and densitometric analysis. Correlational analyses between the SM2-to-SM1 ratio and unloaded shortening in saponin- and alpha-toxin-permeabilized SMCs (n = 28) reveal no significant relationship between the SM-MHC tail isoform ratio and unloaded shortening velocity. The best correlations between SM2/SM1 and the contraction characteristics of untethered vascular SMCs were with the minimum length attained following contraction (n = 20 and r = 0.72 for alpha-toxin, n = 8 and r = 0.78 for saponin). These results suggest that the primary effect of variable expression of the SM1 and SM2 SM-MHC tail isoforms is on the cell final length and not on shortening velocity.

Experimental study of Salvia Miltiorrhiza on the prevention of restenosis after angioplasty

For evaluating the possibility of Salvia miltiorrhiza (SM) on the prevention of arterial restenosis after angioplasty, we investigated the effect of SM on the intimal thickening of air-injured carotid artery of rats. The results showed that the maximal intimal thickness of the injured arteries was much thinner in the treatment group than that in the control group, indicated that SM could prevent experimental restenosis in rat model. Furthermore, we examined the effect of SM on the proliferation of the isolated rabbit arterial smooth muscle cells (SMC) cultivated in vitro. The results showed that SM inhibited the 3H-TdR take-up and proliferation of SMC in a dose-dependent manner. The study raised the possibility that SM could be used to prevent arterial restenosis after angioplasty.

Changes in osteopontin mRNA expression during phenotypic transition of rabbit arterial smooth muscle cells.

Transition from a contractile to a synthetic phenotype appears to be an early key event during the development of intimal thickening after arterial wall injury. We examined the expression of osteopontin mRNA, proliferation, and phenotypic properties of smooth muscle cells (SMCs) in rabbit neointima after balloon denudation and in primary culture. A strong osteopontin mRNA signal was detected in the thickened intima 1 week after balloon denudation and in the surface layer of the intima 2 weeks after balloon denudation. Ki-67 immunohistochemistry showed that osteopontin mRNA expression increased when SMCs entered the proliferating phase in the intima. Rabbit arterial SMCs on type I collagen after 1 day of primary culture with growth factors, as well as freshly isolated cells, were in the G0 phase (contractile phenotype) and did not express osteopontin mRNA. After 3 days of culture, most cells entered the G1B phase (synthetic phenotype) and expressed osteopontin mRNA. In the absence of growth factors, most cells transferred to the G1A phase (intermediate phenotype) after 3 and 7 days, but did not express osteopontin mRNA. Our findings indicate that the osteopontin gene provides a marker that can be used to distinguish the phenotypic properties of vascular SMCs.

Heterogeneity of smooth muscle myosin heavy chain expression at the single cell level.

Expression of the smooth muscle myosin heavy chain (SM-MHC) isoforms was examined in individual rabbit arterial smooth muscle cells with the use of reverse transcription-polymerase chain reactions (RT-PCR). The RT-PCR amplification protocol used oligonucleotide primers complementary to regions that flank the alternative exon that encodes the nine unique amino acids found in the carboxy terminal domain of SM2. RT-PCR products of SM1 and SM2 mRNA differ in length and electrophoretic mobility. Partial DNA sequencing of the PCR products confirmed SM1 and SM2 identity. Densitometric analyses of adjacent samples extracted from the same tissues and processed for SM2-to-SM1 protein and PCR-amplified SM2-to-SM1 RNA ratios exhibited a high correlation (R = 0.92). RT-PCR-amplified SM2-to-SM1 mRNA ratios of individual adult rabbit arterial cells ranged from 0.0 to 1.8 (n = 59), whereas multicellular vascular samples varied much less (0.4-0.6, n = 5). These results indicate that individual cells within a blood vessel differ significantly in SM-MHC expression. This difference may be important for the regulation of contraction in these vessels.

Retardation of Phenotypic Transition of Rabbit Arterial Smooth Muscle Cells in Three-Dimensional Primary Culture

Three-dimensional gel culture systems represent conditions that mimic the differentiated state of mesenchymal cellsin vivo.We examined gel contraction, cell growth, and phenotypic modulation of rabbit arterial SMC in three-dimensional gel culture. The gel contraction rate was dependent on the collagen type; that is, the contraction by freshly isolated SMC was faster and more pronounced in type I collagen than in type III collagen. In contrast, the phenotypic modulation of SMC was independent of collagen type. The major portion of cells in both type I and III collagens with growth factors underwent transition from a contractile (G0 phase) to a synthetic phenotype (G1B phase), but this transition was clearly delayed compared with that on collagens. The cells had hardly begun DNA synthesis in either collagen type and failed to proliferate even after 10 days of culture. These results indicate that collagen type is important in gel contraction by vascular SMC, while the organization of collagen fibrils (two-dimensional vs three-dimensional) is more critical in the phenotypic transition and proliferation of these cells. However, the more specific organization of extracellular matrix than the collagen gel culture system may be necessary to maintain the contractile phenotype of SMC.

Identification of Integrins Involved in Cell Adhesion to Native and Denatured Type I Collagens and the Phenotypic Transition of Rabbit Arterial Smooth Muscle Cells

Rabbit smooth muscle cells (SMC) in primary culture attached to and started proliferating on native and heat-denatured type I collagens, although the amount of cell attachment to denatured collagen was significantly lower. The cells adhered poorly and were unable to grow on commercial gelatin. In contrast, synthetic SMC in secondary culture could adhere to gelatin and grew as well on gelatin as on native type I collagen. The SMC in the contractile state adhered to native type I collagen through the α1β1 and α3β1 integrins. The cells in the intermediate phenotype also adhered to the substrate through the α1β1 and α3β1 integrins, but the relative amount of α3 integrin decreased. The initial adhesion of cells in secondary culture to native type I collagen was mediated only by the α1β1 integrin. The cell-binding sequences did not contain DGEA (Asp-Gly-Glu-Ala) or RGD (Arg-Gly-Asp). In contrast, cell adhesion to heat-denatured type I collagen was mediated only by the α1β1 integrin in the contractile state and by the α1β1, α2β1, and α3β1 integrins in the synthetic state. In heat-denatured type I collagen, the sequences DGEA and RGD served as a recognition site for the α2β1 and α3β1 integrins. Our results suggest that rabbit SMC can recognize the native and denatured type I collagens through interactions with the triple helix-binding receptors and α chain-binding receptors and that the expression pattern of integrins changes in conjunction with the phenotypic properties of vascular SMC.

Changes in Elastin-Binding Proteins during the Phenotypic Transition of Rabbit Arterial Smooth Muscle Cells in Primary Culture

Elastin, a major constituent of the media, may play an important role in the attachment, migration, and phenotypic properties of vascular smooth muscle cells (SMC). We examined binding proteins and binding sites on elastin in rabbit arterial SMC in primary culture. Freshly isolated cells on α-elastin underwent phenotypic transition from a contractile to a synthetic state after 5 days of culture under serum-deficient conditions (1% FBS) and began to proliferate after 6 days of culture (48 h later than on plastic dishes). SMC in the contractile state expressed two binding proteins (130 and 36 kDa), while the 130-kDa band was undetectable in cells in the synthetic state. The adhesion of freshly isolated cells and cells in the synthetic state to α-elastin was significantly inhibited by a monoclonal anti-elastin receptor antibody (BCZ-67). The synthetic peptide VGVAPG (Val-Gly-Val-Ala-Pro-Gly), which contains the recognition sequence for the elastin receptor, inhibited the adhesion of freshly isolated cells to α-elastin at 0.01-1 m M, but showed no inhibitory activity on the adhesion of cells in the synthetic state at 0.01 m M. These findings suggest that α-elastin suppresses the phenotypic transition of rabbit arterial SMC by interacting with the high-molecular-size (130 kDa) binding protein for a cell-binding sequence VGVAPG, while cells in the synthetic state can recognize α-elastin through interactions with the low-molecular-size (36 kDa) binding protein for the sequence VGVAPG.

Cell Adhesion Receptors for Native and Denatured Type I Collagens and Fibronectin in Rabbit Arterial Smooth Muscle Cells in Culture

Cell adhesion molecules serve as specific cell surface receptors for extracellular matrices and contribute to the attachment, spreading, proliferation, and differentiation of vascular cells. We examined the cell adhesion receptors and binding sites on native type I collagen, heat-denatured type 1 collagen, and fibronectin in rabbit arterial smooth muscle cells (SMC) in culture. On fibronectin, anti-α3β1 and anti-α5β1 integrin antibodies and the synthetic peptide GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) significantly inhibited the attachment and spreading of rabbit SMC after 1 and 24 h of culture, while anti-α1β1 inhibited attachment and spreading only after 1 h. In contrast, the attachment and spreading of the cells on native type I collagen were mediated by α1β1 integrin and the cell-binding sequence which did not contain RGD (Arg-Gly-Asp) and DGEA (Asp-Gly-Glu-Ala) after both 1 and 24 h. On heat-denatured type I collagen, α2β1 integrin mediated the cell attachment and spreading after 1 and 24 h and DGEA served as a recognition site for the α2β1 integrin. α1β1 and α3β1 integrins affected only the initial adherence (1 h after plating) of the cells to denatured type I collagen. These findings suggest that rabbit SMC in culture can recognize the native and unfolded triple helical structures of type I collagen by interacting with the collagen fibril-binding receptor (α1β1 integrin) and collagen peptide-binding receptors (α2β1 and α3β1 integrins). Moreover, α1β1 integrin may mediate the initial adherence to each substrate.

Growth Regulation in Primary Culture of Rabbit Arterial Smooth Muscle Cells by Platelet-Derived Growth Factor, Insulin-like Growth Factor-I, and Epidermal Growth Factor

Many studies have linked the proliferation of smooth muscle cells (SMC) to the development of atherosclerotic lesions. We examined the effects of platelet-derived growth factor (PDGF), insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) on the regulation of SMC grown on type I collagen-coated dishes in serum-free primary culture. When added alone, PDGF (10 ng/ml), IGF-I (20 ng/ml), and EGF (10 ng/ml) produced minimal effects on BrdU (5-bromo-2′-deoxyuridine) incorporation into cellular DNA and on cell growth. However, simultaneous addition of PDGF and IGF-I significantly stimulated DNA synthesis and cell growth. The combination of PDGF, IGF-I, and EGF synergistically stimulated DNA synthesis and cell proliferation. Flow cytometric analysis indicated that type I collagen alone promoted the phenotypic modulation and progression of the cells from the G0 (contractile phenotype) to the G1A phase (intermediate phenotype), PDGF and IGF-I, together, stimulated the rate of cell transition from the G1A to the G1B and S phases (synthetic phenotype), and PDGF, IGF-I, and EGF together stimulated the rate of cell transition into the S and G2+M phases. In contrast, in quiescent secondary cultured SMC (G1B phase), PDGF alone was able to initiate DNA synthesis, although IGF-I and EGF were required to complete DNA synthesis. These results reveal that PDGF and IGF-I stimulate the cells to complete the G1A phase and proceed to the G1B phase and that EGF regulates the rate of entry into the S phase in rabbit SMC in primary culture. Furthermore, differences in the responsiveness to these growth factors between primary and secondary cultures reflected the varying phenotypic properties of vascular SMC.

Type I Collagen Promotes Modulation of Cultured Rabbit Arterial Smooth Muscle Cells from a Contractile to a Synthetic Phenotype

The phenotypic transition of smooth muscle cells (SMC) from a contractile to a synthetic state appears to be an early event in the pathogenesis of atherosclerosis. We examined the effects of extracellular matrix components on the phenotypic modulation of rabbit arterial SMC in primary culture by flow cytometry. The results demonstrate that freshly isolated SMC attached, spread, and started to proliferate on type I collagen as well as on fibronectin. Moreover, type I collagen was as efficient as fibronectin in promoting the transition of the cells into the synthetic phenotype without exogenous mitogens. However, unlike on fibronectin, the synthetic peptide GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) and the peptide KDGEA (Lys-Asp-Gly-Glu-Ala), which contains the recognition sequence for α 2β 1 integrin in type I collagen, interfered little with the attachment, spreading, and phenotypic modulation of the cells on type I collagen. On the other hand, the phenotypic modulation of the cells was counteracted by the anti-β 1 integrin antibody. These findings indicate that type I collagen promotes the phenotypic transition of the rabbit arterial SMC by interacting with a cell surface receptor (β 1 integrin family) for a cell-binding sequence without RGD and DGEA. In contrast, elastin, a major constituent of the media, suppressed the cell attachment and spreading and maintained the cells in the contractile phenotype as laminin. These results suggest diverse roles of type I collagen and elastin as well as of fibronectin and laminin in the control of the differentiated properties of arterial SMC.