Bunazosin hydrochloride inhibits exaggerated growth of vascular smooth muscle cells from spontaneously hypertensive rats by suppressing the response to growth factors

Selective alpha1-adrenoreceptor blockers were recently reported to have an in vivo antiproliferative effect on hypertensive cardiovascular organs. Cultured vascular smooth-muscle cells (VSMCs) from spontaneously hypertensive rats (SHRs) show exaggerated growth compared with cells from Wistar-Kyoto (WKY) rats. We investigated the effects of an alpha1-adrenoreceptor blocker, bunazosin hydrochloride (HCl), on the growth of VSMCs from SHRs. In the absence of serum, bunazosin HCl significantly inhibited basal DNA synthesis by VSMCs from SHRs, but not by cells from WKY rats. In the presence of serum, bunazosin HCl significantly inhibited DNA synthesis by VSMCs from both rat strains. Angiotensin (Ang) II, platelet-derived growth factor (PDGF)-AA, and epidermal growth factor (EGF) dose-dependently increased DNA synthesis by VSMCs from SHRs, but not by VSMCs from WKY rats. Bunazosin HCl significantly suppressed the response of DNA synthesis to PDGF-AA and EGF, but not to Ang II, in VSMCs from SHRs. Expression of basic fibroblast growth factor (bFGF), transforming growth factor-beta1 (TGFbeta1), and PDGF messenger RNA (mRNA) was markedly greater in VSMCs from SHRs than in cells from WKY rats. Bunazosin HCl significantly inhibited the expression of bFGF and TGFbeta1 mRNA in VSMCs from SHRs, but not in cells from WKY rats. These findings suggest that the inhibition of growth factor hyperresponsiveness and inhibition of the expression of growth factors in VSMCs from SHRs are associated with the antiproliferative effect of bunazosin.     

Angiotensin II induces hypertrophy of human airway smooth muscle cells: expression of transcription factors and transforming growth factor-beta1

The three-dimensional structure of the complex between a T cell receptor (TCR) beta chain (mouse Vbeta8.2Jbeta2.1Cbeta1) and the superantigen (SAG) staphylococcal enterotoxin C3 (SEC3) has been recently determined to 3.5 resolution. To evaluate the actual contribution of individual SAG residues to stabilizing the beta-SEC3 complex, as well as to investigate the relationship between the affinity of SAGs for TCR and MHC and their ability to activate T cells, we measured the binding of a set of SEC3 and staphylococcal enterotoxin B (SEB) mutants to soluble recombinant TCR beta chain and to the human MHC class II molecule HLA-DR1. Affinities were determined by sedimentation equilibrium and/or surface plasmon detection, while mitogenic potency was assessed using T cells from rearrangement-deficient TCR transgenic mice. We show that there is a clear and simple relationship between the affinity of SAGs for the TCR and their biological activity: the tighter the binding of a particular mutant of SEC3 or SEB to the TCR beta chain, the greater its ability to stimulate T cells. We also find that there is an interplay between TCR-SAG and SAG-MHC interactions in determining mitogenic potency, such that a small increase in the affinity of a SAG for MHC can overcome a large decrease in the SAG's affinity for the TCR. Finally, we observe that those SEC3 residues that make the greatest energetic contribution to stabilizing the beta-SEC3 complex ("hot spot" residues) are strictly conserved among enterotoxins reactive with mouse Vbeta8.2, thereby providing a basis for understanding why SAGs having other residues at these positions show different Vbeta-binding specificities.     

Transforming growth factor-beta1 in hemangioma of the ovary

OBJECTIVE: Anastomotic pseudoaneurysms continue to be a late complication of vascular surgery, particulary following prosthetic graft procedures. The purpose of this study was to investigate if a previously reported increase in interval between the original operation and the development of pseudoaneurysm was related to a change in incidence. DESIGN: Retrospective study. METHODS: We reviewed the records of 76 patients who presented with 90 femoral pseudo-aneurysms and underwent reconstructive procedures from January 1989 to June 1994. The median age was 69 years (range: 39-83). In the same time period all femoral artery anastomosis operations were recorded. RESULTS: The incidence of femoral pseudo-aneurysms in Copenhagen was approximately 4.3%. CONCLUSIONS: A previously reported increase in interval between primary operation and aneurysms formation was not related to a change in incidence during the same time period.     

Transforming growth factor beta1 induces IL-1 receptor antagonist production and gene expression in rat vascular smooth muscle cells

BACKGROUND: We compared long-term results of coronary artery bypass grafting between 1976 and 1988 in 176 patients 40 years old or younger with a matched control group of 176 patients 25 to 30 years older. METHODS: Mean age was 37.4 +/- 2.7 years (+/- standard deviation) in the study group and 64.2 +/- 2.9 years in the control group. Matching criteria were age, sex, left ventricular ejection fraction, number of bypass grafts, and year of operation. RESULTS: The study group had more smokers (p = 0.000) and more patients with hypercholesterolemia (p = 0.026), unstable angina (p = 0.003), and preoperative myocardial infarction (p = 0.009); fewer patients had hypertension (p = 0.000) and diabetes (p = 0.005) in this group than in the control group. The internal mammary artery was used in 31% of the study patients and in 30% of the controls. The actuarial survival rates after 5, 10, and 15 years were 92%, 86%, and 72% in the study group and 92%, 86%, and 66% in the control group (p = 0.202). Young age was a predictor of cardiac reoperation. CONCLUSIONS: Late survival is similar for young and older patients, but the reintervention rate is higher in the younger group. The absence of unstable angina, a left ventricular ejection fraction greater than 0.45, and the use of internal mammary artery grafts increase survival in all patients.     

Interaction between growth factors and kinins in arterial smooth muscle cells

Bradykinin receptors are present on vascular smooth muscle cells; however, the regulation and biological function of these receptors is unclear. To address these questions the interaction between growth factors and kinins in cultured arterial smooth muscle cells has been examined. Based upon the data a hypothesis is presented that platelet-derived growth factor (PDGF) upregulates cell surface bradykinin B2 receptors on arterial smooth muscle cells. The biological effect of the increase in B2 receptors is currently unclear but under certain conditions they may enhance mitogenesis. These mitogenic effects however, are strongly opposed by the effects of bradykinin acting via a B1-type of receptor which mediates potent inhibition of growth factor-induced mitogenesis.     

The effects of perindopril on vascular smooth muscle polyploidy in stroke-prone spontaneously hypertensive rats

OBJECTIVE: To quantify vascular smooth muscle polyploidy and growth kinetics in aortic cells from stroke-prone spontaneously hypertensive rats (SHRSP) and from normotensive Wistar-Kyoto (WKY) rats, and to examine the effects of treatment with the angiotensin converting enzyme (ACE) inhibitor perindopril on these parameters. DESIGN: The following experimental groups were used: young (age < 20 weeks) and old (age > 20 weeks) untreated WKY rats and untreated SHRSP; SHRSP treated with perindopril, and age- and sex-matched control SHRSP; and SHRSP treated with hydralazine and hydrochlorothiazide and age- and sex-matched control SHRSP. The effects of treatment of the SHRSP with perindopril for 30 days on vascular smooth muscle polyploidy and growth kinetics were measured and compared with the effects of equivalent antihypertensive doses of hydralazine and hydrochlorothiazide. METHODS: Vascular smooth muscle polyploidy was measured using flow-cytometry DNA analysis of freshly harvested cells. Growth curves were performed on cultured aortic cells. Plasma renin activity was measured by an antibody-trapping method, plasma angiotensin II (Ang II) by radioimmunoassay and plasma ACE activity by a colorimetric method. Cardiac hypertrophy was evaluated by measuring the heart weight:body weight and left ventricle + septum weight:body weight ratios. RESULTS: The SHRSP had markedly and significantly elevated G2 + M phase of the cell cycle. Treatment with perindopril resulted in a significant reduction in polyploidy in the SHRSP, whereas treatment with hydralazine and hydrochlorothiazide had no effect on the percentage of cells in the G2 + M phase of the cell cycle. The regression of polyploidy after treatment with perindopril was associated with a significant reduction in the concentration of Ang II and ACE activity, and with a significant regression of cardiac hypertrophy. Increased mitogenesis of cultured vascular smooth muscle cells from the SHRSP was not altered by treatment with perindopril. CONCLUSIONS: ACE inhibition reduces vascular smooth muscle polyploidy in large conduit arteries. This type of vascular protection is mediated by the reduced Ang II and possibly by increased kinins level, rather than by the hypotensive effect alone.     

Transforming growth factor-beta in GI neoplasia, wound healing and immune response

The last decade has been marked by tremendous advances in the biochemical and functional characterization of TGF-betas and their receptors in normal and transformed cells. TGF-betas have been shown to modulate proliferation, differentiation and motility of different cell types in a number of in vitro model systems and in some cases with some intriguing results. It is obvious that there is no simple pattern that explains the TGF-betas biological activity in vitro and their effects on cell behaviour need to be assessed in the context of an appropriate physiological cellular environment. Cell-cell and cell-matrix interactions, the differentiating status of the cell together with the functional activity of other soluble growth factors can influence how TGF-betas modulate cell behaviour. However, the overwhelming interest in this field shown by clinicians and basic scientists is rapidly increasing our understanding of how growth factors such as TGF-betas regulate the homeostasis of the GI mucosa and their role in gastrointestinal carcinogenesis.     

Effects of growth rate and cell density on nerve growth factor secretion in cultures of vascular and bladder smooth muscle cells from hypertensive and hyperactive rats

Changes in action potential parameters by and inotropic responses to nicardipine, verapamil, ryanodine and cyclopiazonic acid were examined in isolated ventricular myocardial preparations from neonatal and adult mice. The action potential of both neonatal and adult mice had a unique configuration with little evidence of a plateau at depolarized membrane potential; the action potential duration was significantly larger in neonatal preparations. Nicardipine had no effect on action potential parameters in the adult while it significantly shortened the action potential duration at 50% repolarization in the neonate. Ryanodine significantly shortened the action potential duration at 80% repolarization at both ages: the shortening was significantly larger in the adult when compared with the neonate. The contraction of ventricular preparations from adult mice were relatively resistant to nicardipine and verapamil. Nicardipine or verapamil, even at 10(-5) M, only decreased the contractile force to 70% of control values; the decrease was much less than that reported in other experimental species such as chick, guinea pig or rabbit. In the neonate, 10(-5) M nicardipine or verapamil decreased the contractile force to 30% of control values. Ryanodine had a potent negative inotropic effect both in the neonate and adult; the effect was significantly larger in the adult. Cyclopiazonic acid produced a decrease in contractile force and prolongation of the time required for relaxation; both effects were significantly larger in the adult. These results suggest that the contraction of the adult mouse myocardium is highly dependent on SR function and less dependent on transsarcolemmal Ca2+ influx when compared with the myocardium of the neonatal mouse and that of other species.     

Adenovirus-mediated overexpression of human transforming growth factor-beta 1 in rat cardiac fibroblasts, myocytes and smooth muscle cells

Transforming growth factor-beta 1 (TGF-beta 1) is known to regulate cardiac cell function and its overexpression in the heart is thought to contribute to the development of cardiac hypertrophy and fibrosis. We wished to develop a high efficiency gene transfer method that could be used both in vitro and in vivo and result in the overexpression of TGF-beta 1. For this purpose, we constructed a replication-deficient human adenovirus 5 vector encoding for human TGF-beta 1 and used for control purposes an adenovirus lacZ vector. The adenovirus 5 construct was capable of infecting neonatal rat cardiac myocytes, fibroblasts and VSMCs. Of the three cell types, cardiac myocytes appear more susceptible to infection by the adenovirus 5 construct as assessed through beta-galactosidase staining. Infection of cardiac fibroblasts, myocytes and VSMCs with the hTGF-beta 1 adenovirus leads to the expression of hTGF-beta 1 mRNA and enhanced levels of bioactive and total TGF-beta 1 protein. Infection with hTGF-beta 1 adenovirus also results in enhanced levels of collagen type III gene expression in VSMCs and fibroblasts whereas in cardiac myocytes it leads to increased levels for sarcomeric and beta-actin. Thus, this adenoviral vector might be used for the exploration of in vivo effects of altered levels of cardiac TGF-beta 1.     

Transforming growth factor-beta isoforms in the developing chicken intestine and spleen: increase in transforming growth factor-beta 4 with coccidia infection

Expression of transforming growth factor-betas 2, 3 and 4 (TGF-beta) in the developing chicken intestine and spleen was investigated using specific cDNA probes and antibodies for the different TGF-beta isoforms. Coordinate expression of the mRNAs for TGF-beta s 2, 3 and 4 was detected in the embryonic intestine by 8 days, with maximal expression of the mRNAs for TGF-beta s 2 and 4 occurring at 12 and 19 days, respectively, while expression of TGF-beta 3 mRNA remained constant during this time. While specific antibodies for TGF-beta s 2, 3 and 4 could detect only weak immunohistochemical staining of the intestinal epithelium in 4-, 12- and 16-day-old embryos, intense staining for TGF-beta s 2, 3 and 4 was detected in the tips of the intestinal villi of 19-day-old embryos. In the spleen, expression of the mRNAs for TGF-beta s 2 and 3 increased in the newly hatched chick compared with the embryo and then decreased in the adult to levels that were lower than in the embryo; expression of TGF-beta 4 mRNA increased progressively with developmental age, with expression in the adult spleen being significantly higher than in the embryonic and hatchling spleen. Immunohistochemical staining of spleens showed a selective increase in the level of reactive TGF-beta 4 with increasing developmental age, while staining for TGF-beta s 2 and 3 was constant during development. After infection of 1-month-old chickens with coccidian parasite, expression of TGF-beta 4 mRNAs increased 5-8-fold in intestinal intra-epithelial lymphocytes and 2.5-fold in spleen cells, while expression of the mRNAs for TGF-beta s 2 and 3 remained constant in these cells. The results of this study suggest that TGF-beta may play a role in development of the intestine and spleen in the chicken and that TGF-beta 4 in particular increases after infection of coccidia in the chicken.     

Single L-type calcium channels in smooth muscle cells from resistance arteries of spontaneously hypertensive rats

The amplitude of the whole-cell L-type Ca2+ channel current recorded from vascular smooth muscle cells is reportedly greater in spontaneously hypertensive rats (SHR) than in Wistar-Kyoto rats (WKY). However, no study has examined properties of single Ca2+ channels in arterial cells from these strains. To further test the hypothesis that activation of L-type Ca2+ channels in arterial smooth muscle cells would be enhanced in SHR, we recorded single Ca2+ channel currents in resistance mesenteric artery cells from SHR and WKY (8 to 9 weeks of age) using a cell-attached patch clamp technique. With 50 mmol/L Ba2+ in the recording pipette, the depolarizing pulse from a holding potential of -40 mV evoked the single L-type Ca2+ channel current. Opening of the single channels was more frequent in cells from SHR than from WKY. Single-channel conductance (20 pS) and open time (1 ms at 0 mV) did not differ in the two strains. The results suggest that an increased amplitude of the whole-cell current can be attributed to the enhanced opening of single Ca2+ channels in the arterial smooth muscle cells from SHR compared with WKY.     

Differential expression of the keratinocyte growth factor (KGF) and KGF receptor genes in human vascular smooth muscle cells and arteries

The objective of this study was to analyze the efficacy and correlation between clinical and histologic parameters used to evaluate oral implants. After extraction of the premolars and a healing time of 4 months in 16 Dutch goats, four Br?nemark implants were placed in the maxillary left and right premolar regions. After a healing time of 6 months, followed by another 4 months with the permucosal abutments, the goats were sacrificed and the jaws were block-resected. Before histologic preparation, long-cone radiographs were made and Periotest scores of the implants were recorded. Bone level measured histomorphometrically were found to be 0.85 mm more apically, compared to that measured radiologically (P = .001). Furthermore, statistically significant correlations (P > 0.2) were not found between the Periotest values of the calcium-phosphate-coated and uncoated implants for (1) the first thread in contact with bone, or (2) with the total number of threads in contact with bone. It was concluded that the radiologic data overscored the real marginal bone level around screw-shaped oral implants, and that the Periotest device is neither able to discriminate between the first thread nor between the total number of threads in contact with bone.     

Different calcium storage pools in vascular smooth muscle cells from spontaneously hypertensive and normotensive Wistar-Kyoto rats

OBJECTIVE: To evaluate whether the distribution of intracellular free calcium may be impaired in primary hypertension. DESIGN: Cytosolic free calcium and stored calcium were investigated in cultured vascular smooth muscle cells from spontaneously hypertensive rats (SHR). METHODS: The concentrations of intracellular and stored calcium were investigated in cultured vascular smooth muscle cells from spontaneously hypertensive rats aged 6 months from the Münster strain (SHR) and from age-matched normotensive Wistar-Kyoto (WKY) rats. Vascular smooth muscle cells were grown on coverslips, and fluorescence measurements of the intracellular calcium concentration were performed using fura-2. The different effects of thapsigargin, a selective Ca-ATPase inhibitor, and of angiotensin II (Ang II) on the calcium storage pools were investigated. RESULTS: In the absence of external calcium thapsigargin produced a dose-dependent transient increase in the concentration of intracellular calcium in vascular smooth muscle cells. The thapsigargin-induced maximum peak increase in the concentration of intracellular calcium was not significantly different in SHR and WKY rats. After depletion of the thapsigargin-sensitive calcium pools the addition of 100 nmol/l Ang II produced a rise in the concentration of intracellular calcium in vascular smooth muscle cells from SHR and WKY rats. Using vascular smooth muscle cells from the SHR the Ang II-induced increase in the concentration of intracellular calcium was not significantly different in the presence and absence of thapsigargin, indicating that the calcium pools depleted by thapsigargin and Ang II do not overlap significantly in vascular smooth muscle cells from SHR. In contrast, in the WKY rats the response to Ang II was significantly diminished after depletion of the thapsigargin-sensitive pool. When Ang II and thapsigargin were administered in the reverse order, i.e. Ang II before thapsigargin, the thapsigargin response was diminished in the WKY rats but not in the SHR. CONCLUSION: SHR differ from WKY rats in having vascular smooth muscle cells that contain thapsigargin-sensitive calcium storage pools that are distinct from the Ang II-sensitive calcium pools.     

Low-affinity thromboxane receptor mediates proliferation in cultured vascular smooth muscle cells of rats

We examined the binding properties and mitogenic effects of U46619, using cultured vascular smooth muscle cells (VSMCs), by ligand-binding assay, measuring [3H]thymidine and [3H]leucine incorporation, checking with flow cytometry, and counting the cell number. The U46619-activated mitogenic signal-transduction pathway was assessed by measuring formation of inositol monophosphate (IP); [Ca2+]i; mitogen-activated protein kinase (MAPK), MAPK kinase (MAPKK), and p74raf-1 activities; and GTP-bound Ras. [3H]U46619 bound to cultured VSMCs from Wistar-Kyoto (WKY) rats at a single class of site (Kd: 15.5 +/- 2.6 nmol/L). However, it bound to VSMCs from spontaneously hypertensive rats (SHRs) at two classes of sites (Kd: 2.3 +/- 0.6 nmol/L and 1.4 +/- 0.5 mumol/L). U46619 increased DNA and protein synthesis, cell number, IP formation, [Ca2+]i, and MAPK and MAPKK activities, with EC50 values close to its Kd value for the low-affinity binding site in VSMCs from SHR. Prostaglandin (PG) E2 and PGF2 alpha showed little of such mitogenic effects. All these effects of U46619 were inhibited by SQ29548, staurosporine, or pretreatment of VSMCs with phorbol 12-myristate 13-acetate for 24 hours. However, U46619 stimulation did not lead to a significant increase in the Ras-GTP complex or p74raf-1 activity. In conclusion, the mitogenic effect of U46619 appears to be mediated via the activation of low-affinity thromboxane binding sites that trigger phosphoinositide hydrolysis and activate the MAPK pathway, leading to DNA synthesis and cell proliferation.