Proliferation kinetics of aortic smooth muscle cell populations: comparison of normotensive and spontaneously hypertensive rats

Abstract. An in vitro autoradiographic study of the proliferation of smooth muscle cells (SMC) from the aorta of normotensive and spontaneously hypertensive rats has been made. It was found, in primary culture, that SMC of spontaneously hypertensive rats entered proliferation at 2-2.5 days later than those from normotensive animals. As revealed by their very intensive labelling, a subpopulation of SMC with a high turnover rate was found in primary culture. In freshly isolated SMC from normotensive rat aorta, a subpopulation in S phase was detected, but we failed to detect it in aortae from spontaneously hypertensive rats. A difference in proliferative behaviour was also observed in subcultures of SMC from rats of both strains.     

Proliferation kinetics of aortic smooth muscle cell populations: comparison of normotensive and spontaneously hypertensive rats

An in vitro autoradiographic study of the proliferation of smooth muscle cells (SMC) from the aorta of normotensive and spontaneously hypertensive rats has been made. It was found, in primary culture, that SMC of spontaneously hypertensive rats entered proliferation at 2-2.5 days later than those from normotensive animals. As revealed by their very intensive labelling, a subpopulation of SMC with a high turnover rate was found in primary culture. In freshly isolated SMC from normotensive rat aorta, a subpopulation in S phase was detected, but we failed to detect it in aortae from spontaneously hypertensive rats. A difference in proliferative behaviour was also observed in subcultures of SMC from rats of both strains.     

Enhanced growth-dependent expression of TGF beta 1 and hsp70 genes in aortic smooth muscle cells from spontaneously hypertensive rats.

Enhanced proliferation of vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) as compared with Wistar-Kyoto rats (WKY) persists in long-term culture and is characterized by an accelerated entry of these cells into the synthetic S phase of the cell cycle and a higher specific growth rate, particularly evident at high cell density. In the present study, we investigated by Northern blot experiments the expression of genes putatively involved in the regulation of VSMC growth. One of them is the transforming growth factor beta 1 gene (TGF beta 1), a bifunctional modulator of cell growth whose action is dependent on cell density. The accumulation of TGF beta 1 mRNA was enhanced in growing SHR cells at every density studied as early as 24 h after inoculation with a further increase at later times. Protooncogenes c-fos and c-myc, which have been implicated in G1/S phase transition, have also been investigated in VSMC by Northern blot analysis. At low cell density, calf serum stimulated c-fos and c-myc mRNA expression was comparable in WKY and SHR cells whereas at high cell density, c-fos induction was higher in VSMC from SHR. SHR VSMC respond more to mitogenic stimulation and to environmental (e.g., heat) stress, particularly when growing near saturation density. hsp70 constitutes a gene family responsive to environmental stimuli (heat) and to mitogenic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet-derived growth factor AA homodimer stimulates protein synthesis rather than DNA synthesis in vascular smooth muscle cells from spontaneously hypertensive rats but not from normotensive rats.

Platelet-derived growth factor (PDGF) AB and BB isoforms were potent mitogens for cultured vascular smooth muscle cells from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). PDGF-AA promotes protein synthesis in a dose-dependent manner in SHR cells, whereas DNA synthesis was stimulated only slightly. However, this isoform did not activate either DNA or protein synthesis in WKY cells. PDGF-AA stimulated tyrosine phosphorylation of its receptor protein and phospholipase C-gamma 1 in SHR cell but not in WKY cells. These results indicate that vascular smooth muscle cell of SHR is uniquely responsive to PDGF-AA, presumably due to abnormality in receptor expression, in its hypertrophic response.     

Increase in 12-lipoxygenase activity in platelets of spontaneously hypertensive rats

12-Lipoxygenase activity in platelets of spontaneously hypertensive rats was investigated. Enzyme activity was measured in the absence and the presence of reduced glutathione. In both assay conditions, 12-lipoxygenase activity in platelets of spontaneously hypertensive rats was significantly higher than that in platelets of normotensive rats. Since 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), a 12-lipoxygenase product of arachidonic acid in platelets, has been reported to be a potent chemoattractant for aortic smooth muscle cells, increase in biosynthesis of 12-HETE in platelets of spontaneously hypertensive rats might contribute to the explanation of pathogenesis of vascular disorder commonly found in hypertension patients.     

Isolation and characterization of clonal vascular smooth muscle cell lines from spontaneously hypertensive and normotensive rat aortas

Summary Vascular smooth muscle cells were isolated from the aortas of spontaneously hypertensive rats and normotensive Wistar-Kyoto rats by use of the explant method on collagen gels. Clonal cell lines derived from these enriched populations possessed ultrastructural characteristics of vascular smooth muscle cells in culture; they grew in hill and valley configuration, immunostained with the muscle actin antibody HHF35, and failed to react with von Willebrand Factor VIII antibody. Fourteen clonal cell lines were characterized for growth and ligand binding characteristics. Large variations in growth rate and cell density at saturation were exhibited by clones of both strains. Similar variability was noted for specific binding of endothelial 1 and Sar¹,Ile⁸-angiotensin II to their receptors, indicating considerable phenotypic heterogeneity among the clonal cell lines. Six selected clones were further characterized for angiotensin II receptor linkage to G proteins. Cells of both strains exhibited comparable affinity shifts in the presence of GTPγS. These clonal cell lines should be useful for a variety of analyses of the comparative biology of aortic cells. It is possible that the diversity of phenotypic traits exhibited by these clones reflects the heterogeneity of vascular smooth muscle tissue found in vivo.     

The elevation of the cytoplasmic calcium ions in vascular smooth muscle cells in SHR--measurement of the free calcium ions in single living cells by lasermicrofluorospectrometry

We have developed an accurate and sensitive system for the measurement of cytoplasmic free calcium concentrations ([Ca++]i) of a single cell by using UV-laser and Indo-1. By this method, we made the first successful measurement of [Ca++]i of single living vascular smooth muscle cells. [Ca++]i in spontaneously hypertensive rats was elevated and maintained after the 6th passage culture. However, [Ca++]i in Goldblatt hypertensive rats was not elevated. Thus, these results suggest that the maintenance of high [Ca++]i levels of vascular smooth muscle cells in spontaneously hypertensive rats is genetically regulated and that it is one of the mechanisms for hypertension.     

Differential stimulation of growth related metabolism in cultured smooth muscle cells from SHR and WKY rats by combinations of EGF and LDL

We have reported previously that vascular smooth muscle cells from spontaneously hypertensive rats (SHR) were more responsive to epidermal growth factor (EGF) than their normotensive derived Wistar Kyoto (WKY) controls. This differential responsiveness is evident for several cellular processes including activation of S6-kinase, elevation of intracellular pH and stimulation of both phosphoinositide metabolism and DNA synthesis. Quiescent smooth muscle cells exposed to low density lipoprotein (LDL) exhibited a similar differential responsiveness (SHR greater than WKY) in terms of S6-kinase activation, which was time- and dose-dependent (10(-10)-10(7) M), but neither cell type responded appreciably to LDL in terms of a stimulation in [3H]-thymidine incorporation. Exposure of the same cells to EGF and LDL in combination elicited a marked synergistic stimulation in DNA synthesis, the extent of which was greater for SHR than WKY. The sensitivity of both cell types to EGF was increased in the presence of LDL, although cells from hypertensive animals still exhibited their greater (vs. WKY) sensitivity. In both cell types, activation of nuclear protooncogenes c-fos and c-myc by LDL was minimal, whereas oncogene induction by EGF was approximately five-fold greater for SHR-derived cells compared to those from WKY animals. No marked synergistic effect on the time-dependent induction of either entity was observed for cells exposed to EGF and LDL simultaneously, and the response of SHR-cells remained greater than WKY-cells.     

Specific growth stimulation of cultured smooth muscle cells from spontaneously hypertensive rats by platelet-derived growth factor A-chain homodimer.

Cultured vascular smooth muscle cells (VSMC)1 from spontaneously hypertensive rats (SHR) possess specific cell surface receptors for both homodimeric forms of platelet-derived growth factor (PDGF-AA and PDGF-BB), in contrast to cells from normotensive Wistar Kyoto (WKY) animals, which express receptors only for the B-chain form of PDGF. Stimulation of quiescent VSMC from SHR with PDGF-AA resulted in activation of S6-kinase and induction of phosphoinositide catabolism, as well as cellular proliferation when cultures were maintained for prolonged periods with daily supplementation of the growth factor. WKY-derived VSMC showed no response to PDGF-AA, which was consistent with their lack of specific receptors for this homodimer. The responsiveness of quiescent cells from SHR and WKY to the B-chain homodimer was similar. The enhanced growth responsiveness of SHR-derived cells to fetal calf serum, as compared with cells from their normotensive counterparts, may be accounted for in part by their expression of receptors for the AA homodimer of PDGF.     

CL 115,347 (DHV-PGE2 ME): a new orally and topically active prostaglandin antihypertensive agent

CL 115,347 orally (0.25-10 mg/kg) and topically (0.03 and 0.1 mg/kg) lowered blood pressure in a dose-dependent manner in conscious spontaneously hypertensive rats (SHR). Duration of action of the oral dose range was from 1 to more than 8 h and of the topical dose range, from more than 6 to more than 24 h. CL 115,347 was 100-200 times more potent orally and greater than 250 times more potent topically than l-prostaglandin (PG) E2. When 3 mg/kg was administered orally, CL 115,347 was also active in Dahl "S" salt-sensitive hypertensive rats, deoxycorticosterone acetate-salt hypertensive rats, aorta-coarcted renin-dependent hypertensive rats, normotensive rats, bilaterally nephrectomized SHR, and bilaterally ureteral-ligated SHR. CL 115,347 was also orally active at 0.1 mg/kg in normotensive rhesus monkeys and in renal hypertensive dogs at 1 mg/kg. CL 115,347 was as active as l-PGE2 in relaxing the rabbit ear arterial smooth muscle in vitro. In anesthetized dogs, CL 115,347 injected intra-arterially (0.5-10 micrograms) into the vascular bed being studied increased blood flow to femoral, carotid, coronary, superior mesenteric, and renal vascular beds. CL 115,347 decreased vasopressor responses induced by electrical stimulation of the spinal cord at T7-T9 but did not decrease the tachycardia induced by stimulation of the cardioaccelerator segments (C7-T1) in pithed SHR. CL 115,347 has a broad spectrum of antihypertensive activity in various animal models and probably exerts its major antihypertensive effects through relaxation of blood vessels.     

Expression of smooth muscle MyHC B in blood vessels of hypertrophied heart in experimentally hypertensive rats

We demonstrated recently a significantly lower fraction of cardiac precapillary arterioles that expressed smooth muscle myosin heavy chain (MyHC) B (SMB) in spontaneously hypertensive rats. To clarify whether this reduction of SMB expression is of genetic origin, we investigated SMB expression in cardiac precapillary arterioles of normotensive and experimentally hypertensive rats (one clip, one kidney or ANG II minipump). We observed similar SMB expression patterns in precapillary arterioles of experimentally hypertensive rats compared with normotensive controls. These observations suggest that the downregulation of SMB in spontaneously hypertensive rats is of genetic origin rather than an adaptive response to chronically enhanced blood pressure and cardiac hypertrophy.     

Effect of emotional stress on the contractility, adrenoreactivity and calcium sensitivity of smooth muscles of the portal vein in spontaneously hypertensive rats

The emotional stress decreased contractility and adrenoreactivity in normotensive rats, while in spontaneously hypertensive rats (SHR) the changes were less pronounced. The changes in adrenoreactivity, as well as increased permeability of smooth muscle cells for calcium and reduced reactivity to exogenous calcium revealed in hypertensive animals contribute to high resistance of portal vein smooth muscles to stress.     

The Roles of Protein Kinase C βI and βII in Vascular Smooth Muscle Cell Proliferation

The role of protein kinase C (PKC) on proliferation of A10 vascular smooth muscle cells (VSMC) was studied by overexpressing specific PKC-βI and -βII isozymes. PKC-βI and -βII are derived from alternative splicing of the exon encoding the carboxy-terminal (C-terminal) 50 or 52 amino acids, respectively. The differential functions of the two isozymes with regard to cell proliferation, DNA synthesis, and the cell cycle were investigated in A10 cells, a clonal cell line of VSMC from rat aorta, and in A10 cells overexpressing PKC-βI and PKC-βII (βI-A10 and βII-A10). PKC levels were increased three- to fourfold in heterogeneous cultures of stably transfected cells. Although doubling time of A10 cells was 36 h, the cell doubling time in βI-A10 cells decreased by 12 h, and, in contrast, the doubling time of βII-A10 cells increased by 12 h compared to A10 cells. The increase of [³H]thymidine (TdR) incorporation was accelerated and increased in βI-A10 cells, but slowed and diminished in βII-A10 cells compared to A10 and control cells transfected with empty vector. Cell cycle analysis of βI-A10 cells showed an acceleration of S phase entry while βII-A10 cells slowed S phase entry. These results suggest that PKC-βI and PKC-βII regulate cell proliferation bidirectionally and that PKC-βI and PKC-βII may have distinct and opposing functions as cell cycle check point mediators during late G₁phase and may regulate S phase entry in A10 VSMC.     

Elastase-like enzyme in the aorta of spontaneously hypertensive rats

In an attempt to obtain information regarding vascular elastase in arterial hypertension, we examined biochemical changes in elastase-like enzyme activity, and the intravascular localization of elastase by immunohistochemical techniques in the aorta of spontaneously hypertensive rats (SHR). In the biochemical study, aortic elastase-like enzyme activity was significantly higher in SHR than in controls. Using an antibody against rat pancreatic elastase raised in the rabbit, it was demonstrated immunohistochemically that the enzyme was localized in the endothelial cells and subendothelial spaces in the aorta of control animals. In SHR, elastase was also demonstrated in medial smooth muscle cells and particularly in the modified smooth muscle cells in areas of intimal thickening. Some vacuoles in the smooth muscle cells also showed positive enzyme staining. Elastase seems to play an important role in the development of hypertensive vascular changes.     

Incidence of multinucleated and polyploid aortic smooth muscle cells cultured from different age groups of spontaneously hypertensive rats.

Cell size and incidence of multinucleated, polyploid cells in cultured aortic smooth muscle cells from different age groups of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) were compared. Smooth muscle cells from SHR were generally larger than those from WKY, and the percentage of multinucleated smooth muscle cells was always higher in SHR than WKY in the three age groups of rats studied (3-4, 10-12, and 28-30 weeks). In smooth muscle cells from the 3- to 4-week group, there was a positive correlation between cell diameter and the percentage of multinucleated smooth muscle cells. Microdensitometric measurements also showed that the incidence of polyploid smooth muscle cells was always higher in SHR than WKY in the three age groups. There was a positive correlation between DNA density and nuclear area measurements in all the age groups of SHR and WKY. We conclude that cultured aortic smooth muscle cells from different age groups of SHR and WKY contained heterogeneous populations of cells and that, under our culture conditions, the polyploidy of the smooth muscle cells found in vivo was maintained in the SHR and WKY.     

Polyamine synthesis inhibition induces S phase cell cycle arrest in vascular smooth muscle cells

Polyamines are important for cell growth and proliferation and they are formed from arginine and ornithine via arginase and ornithine decarboxylase (ODC). Arginine may alternatively be metabolised to NO via NO synthase. Here we study if vascular smooth muscle cell proliferation can be reversed by polyamine synthesis inhibitors and investigate their mechanism of action. Cell proliferation was assessed in cultured vascular smooth muscle A7r5 cells and in endothelium-denuded rat arterial rings by measuring [³H]-thymidine incorporation and by cell counting. Cell cycle phase distribution was determined by flow cytometry and polyamines by HPLC. Protein expression was determined by Western blotting. The ODC inhibitor DFMO (1–10 mM) reduced polyamine concentration and attenuated proliferation in A7r5 cells and rat tail artery. DFMO accumulated cells in S phase of the cell cycle and reduced cyclin A expression. DFMO had no effect on cell viability and apoptosis as assessed by fluorescence microscopy. Polyamine concentration and cellular proliferation were not affected by the arginase inhibitor NOHA (100–200 μM) and the NO synthase inhibitor l-NAME (100 μM). Lack of effect of NOHA was reflected by absence of arginase expression. Polyamine synthesis inhibition attenuates vascular smooth muscle cell proliferation by reducing DNA synthesis and accumulation of cells in S phase, and may be a useful approach to prevent vascular smooth muscle cell proliferation in cardiovascular diseases.     

Dependence of the number of vesicles in vascular smooth muscle cells on the presence of noradrenaline in the extracellular space

Noradrenaline (NA) effect on the number of vesicles in smooth muscle cells was investigated in small mesenteric arteries of spontaneously hypertensive rats (SHR), aged 8 or 12 weeks, and age-matched normotensive Wistar-Kyoto (WKY) rats. The presence of NA in the incubation medium resulted in an increase in the number of vesicles in SHR of both age groups, but not in WKY. The results are discussed in view of the relationship between the vesicles and Ca transport in smooth muscle cells.     

Changes of beta-adrenoceptors in the aortic muscle cells of spontaneously hypertensive rats

The characteristics of [125I]monoiodocyanopindolol (ICYP) binding to beta-adrenoceptors of cultured aortic smooth muscle cells derived from 4-week-old spontaneously hypertensive rats (SHR) and the Wistar-Kyoto normotensive rats (WKY) were examined. During optimization of the binding assays, we found that the specific binding of ICYP by intact cells was masked by a high level of nonspecific ICYP accumulation in intact cells presumably owing to the lipophilic nature of ICYP. Optimal specific ICYP binding requires that the cells be gently lysed with hypotonic dilution followed by a freeze-and-thaw cycle. Under most experimental conditions tested, the total number of ICYP binding sites in WKY aortic muscle cells was considerably and consistently smaller than that in SHR cells. There was no difference in the Kd values for ICYP binding to SHR and WKY cells. However, when ICYP binding was carried out using crude membrane fractions with well-defined plasma membrane content isolated from aortic muscle strips of adult rats, we found no difference in the number of beta-adrenoceptor sites between SHR and WKY. Morphological evidence indicated that cultured SHR aortic muscle cells contained a greater proportion of larger cells with multinuclear features. These results suggest that an increase in the number of beta-adrenoceptor density per cell in SHR may be associated with cellular hypertrophy of aortic smooth muscle cells. We conclude that under cultured conditions, a higher incidence of polyploid smooth muscle cells in the SHR as compared with WKY was expressed earlier than under in vivo conditions. Therefore, the interpretation of results obtained from cultured cell studies in relation to under in vivo conditions should be exercised with caution.