Isolation and culture of adult and fetal rabbit bladder smooth muscle cells and their interaction with biopolymers

Purpose: The aim of this study was to test the feasibility of isolation and culture of adult and fetal rabbit bladder smooth muscle cells (SMCs) and comparison of their interactions with different types of biodegradable biopolymers in cell culture. Methods: Bladder SMCs isolated from adult and fetus rabbits were identified by immunostaining for smooth muscle [alpha ]-actin and myosin. Growth kinetics of SMCs were estimated using population doubling time (PDT) and thymidine labeling index (TLI). Poly (D, L-lactide-co-glycolide; PLGA) copolymers were synthesized at 85:15 and 75:25 monomer ratios. The porous scaffolds prepared from these polymers were seeded with SMCs. The study compared the effectiveness of adsorbing fibronectin and fetal calf serum (FCS) on these biopolymers. The cells grown on these polymers were quantified using a neutral red uptake assay. Results: Over 90% of the 2 cell populations stained positive for SMC marker proteins. Fetal SMCs were seen to emerge from the tissue after 3 to 4 days, whereas adult SMCs were seen after 5 to 6 days. However, estimated PDT of fetal and adult SMCs was 85.2 and 54.5 hours, respectively, and TLI of adult SMCs was also higher than with fetal SMCs. Proliferation on 75:25 PLGA was better than for 85:15 and for both biopolymers; adsorption of FCS significantly affected cell attachment relative to fibronectin. Conclusions: Although fetal SMCs were shown to emerge from explants early after seeding onto dishes, doubling time and S-phase fraction of adult bladder SMCs were markedly higher than of fetal derived cells. Adsorption of serum proteins significantly enhances the attachment of both fetal and adult SMCs to biopolymers. J Pediatr Surg 38:21-24.     

Contribution of proliferating leukocytes to phenotypic change in smooth muscle cells during the development of coronary arteriosclerosis in transplanted hearts

BACKGROUND: It is well known that coronary arteriosclerosis after heart transplantation is concentric and rich in smooth muscle cells (SMCs); however, the role played by rejection in the intimal thickening caused by SMCs in coronary arteriosclerosis remains unclear. In this study, we examined the process of intimal hyperplasia caused by SMCs and evaluated the relationship between the differentiation state of SMCs and local inflammation caused by rejection. METHODS: Lewis rat hearts were heterotopically transplanted into F344 rats (allotransplantation group) or other Lewis rats (isotransplantation group). Cyclosporin A (5 mg/kg/day) was injected intramuscularly for 20 days after transplantation in both groups. The transplanted hearts were examined immunohistochemically using several monoclonal antibodies; namely, HHF-35, CGA7, vimentin, alpha-actin, HIS36, R73 and proliferating cell nuclear antigen (PCNA). To evaluate the degree of local immunological response caused by rejection, the anti-PCNA antibody was used. To reveal the subtypes of proliferating cells in the thickened intima, HIS36 and R73 antibodies were used. RESULTS: In the allotransplantation group, SMCs in the media began to undergo a phenotypic change toward a poorly differentiated state 30 days after transplantation. Intimal hyperplasia was observed 60 days after transplantation, the thickened intima being composed mainly of dedifferentiated SMCs with abundant PCNA(+), most of which were macrophages and T cells. The state of differentiation of SMCs in the thickened intima 90 days after transplantation varied from a dedifferentiated to a highly differentiated state. These changes were strongly correlated with the expression of PCNA. CONCLUSION: The expression of PCNA was strongly correlated with the differentiation state of SMCs. Thus, local inflammation caused by rejection may play an important role in the initiation of phenotypic change in SMCs.     

Chronic high pressure potentiates the antiproliferative effect and abolishes contractile phenotypic changes caused by endothelial cells in cocultured smooth muscle cells

High in vitro pressures have been reported to alter smooth muscle cell (SMC) and endothelial cell (EC) phenotype, while endothelial cells (ECs) can influence the proliferation, phenotype, and contractile features of smooth muscle cells (SMC) in coculture systems. However, little is known about the in vitro effects of pressure on EC/SMC cocultures. We therefore sought to compare SMC proliferation in independent and EC coculture under ambient and high pressure, and identify changes in the contractile phenotype of SMCs by measuring levels of the L-type Ca(2+) channel a(1) subunit (dihydropyridine-DHP receptor) which is critical for Ca(2+) transients, differentiation and contractility in SMC. METHODS: Rat aortic SMCs in independent culture (SMC/0) and coculture with ECs (SMC/EC) were maintained in 5% CO(2) under either atmospheric or high pressure (130 mmHg). SMC were counted at 0, 1, 3, and 5 days and compared to initial cell counts of day 0 before the exposure to experimental conditions. DHP receptor levels were quantitated by Western blotting (three similar studies). RESULTS: ECs suppressed SMC proliferation on day 1 of coculture in both atmospheric and high pressure (20% inhibition vs independent culture, P < or = 0.05). By day 3, cocultured SMC under atmospheric pressure displayed no EC-mediated inhibition, and at day 5, atmospheric cocultured SMCs revealed statistically significant enhanced proliferation as compared with SMCs in independent cultures. However, cocultured SMCs exposed to 130 mmHg pressure displayed sustained sensitivity to EC growth inhibition at both days 3 and 5 of the experiment. Coculture decreased SMC DHP-receptor levels under atmospheric pressure. However, this effect was abolished in cocultures exposed to high pressure. CONCLUSIONS: High pressure substantially alters the regulatory influence of EC on SMC proliferation and contractile potential. This pressure/coculture model should increase our understanding of cellular interaction in hypertensive vasculopathy.     

PDGF-BB和TGF-β1诱导人脂肪基质干细胞向血管平滑肌细胞的分化

目的：探讨人脂肪基质干细胞体外诱导为血管平滑肌细胞的可能性。方法：应用酶消化法从人脂肪组织获得单个核细胞,基础培养液培养,经传代后,第一代细胞用于诱导分化实验。未诱导组培养液为基础培养液,诱导组培养液在基础培养液内添加诱导因子PDGF-BB（50ng/mL）、TGF-β1（5ng/mL）,诱导14d后,光镜下观察诱导细胞形态学的改变;免疫荧光和RT-PCR方法检测平滑肌细胞特异标记的表达情况;流式细胞仪检测诱导阳性率。结果：脂肪基质干细胞在特定细胞因子诱导作用下,细胞生长呈现与平滑肌细胞类似的“峰-谷”生长模式,并表达α-SM-Actin、SM-MHC、Calponin、SM-22α等平滑肌特异性标记;诱导组阳性率与未诱导组差异有统计学意义（P〈0.01）。结论：PDGF-BB、TGF-β1可诱导人脂肪基质干细胞向血管平滑肌细胞表型分化。     

Human prostatic smooth muscle cells in culture: Estradiol enhances expression of smooth muscle cell-specific markers

Smooth muscle cells (SMCs) constitute a major cellular component of prostatic stroma. SMC tension plays an important role in urethral obstruction secondary to benign prostatic hyperplasia (BPH). We have developed an in vitro procedure for the propagation of human prostatic SMCs. Tissue specimens from patients undergoing radical prostatectomy or cystectomy were enzymatically disaggregated and cultured in MCDB-131 medium supplemented with horse serum, insulin, conditioned medium from the tumor cell line CRL-5813, and steroid hormones. The medium was assembled on the basis of the effects these supplements have on the growth of SMC cultures and on the expression of the two markers desmin and smooth muscle myosin. Addition of 0.1 μM of estradiol to the growth medium dramatically increased expression of these SMC-specific markers. Dihydrotestosterone (DHT) and hydrocortisone had a similar, albeit less pronounced effect. At three to five passages, about two thirds of the cells were immunohistologically positive for smooth muscle myosin or desmin. Almost all cells were positive for the myofibroblast marker smooth muscle α-actin throughout 10 passages and more. In SMC cultures, cells staining for smooth muscle myosin and desmin were found to seek direct contact to myofibroblasts. They grew in aggregates on a layer of myofibroblasts which adhered to the surface of the culture vessel. As revealed by transmission electron microscopy the cultured cells exhibited morphological features of myofibroblasts. Characteristics of smooth muscle cells, such as prominent bundles of microfilaments associated with dense bodies, basal laminae investing the cells, and numerous caveolae at the cell surfaces were regularly observed in cultures of low passages. After several passages, these features were markedly decreased and organelles of the biosynthetic system became more prominent. In summary, we present an in vitro model of prostatic SMCs and demonstrate that steroid hormones have characteristic effects on these cells. SMC cultures are expected to facilitate investigation of the functions and properties of human prostatic SMCs. Prostate 30:117–129, 1997. © 1997 Wiley-Liss, Inc.     

Performance of a Modified Rabbit Model of Abdominal Aortic Aneurysm Induced by Topical Application of Porcine Elastase: 5-Month Follow-up Study

ObjectivesTo modify the method for creating an abdominal aortic aneurysm in rabbits, and to study its performance.Materials and methodsA total of 24 New Zealand white rabbits were induced topically with 10 μl of porcine elastase (0, 0.1, 5 and 10 units μl^?1) to define the optimal concentration (groups A–D). Twelve aneurysms were induced with 10 units μl^?1 of 10 μl elastase to serve as a follow-up group (group E) to serve as a follow-up. A 1.5-cm aortic segment was isolated and induced with elastase solution for 30 min.ResultsAll animals in groups D and E developed AAA by day 5. Aneurysms in Group E were stable over 100 days. Partial destruction to disappearance of elastic lamellae and smooth muscle cells (SMCs) was seen in elastase-treated animals by day 5. Regenerated elastin and proliferated SMCs were present in group E. Matrix metalloproteinases 2 and 9 and RAM11 showed strong expression in group D, but expression decreased in group E after day 15.ConclusionsThe rabbit AAA model induced via topical application of porcine elastase at 10 units μl^?1 for 30 min appears easy and simple, with shorter induction and more rapid aortic dilation. The model is stable over 100 days and is useful to study the formation and progress of AAAs.     

Characterization of neuropathic bladder smooth muscle cells in culture

PURPOSE: Clinically bladder cells used in tissue engineering techniques will come from neuropathic bladders and not normal bladders. We determined if neuropathic bladder smooth muscle (SM) cells (SMCs) retain functional differences when cultured in vitro. MATERIALS AND METHODS: Primary cultures of SMCs were established from patients with a neuropathic bladder (5) and a normal bladder (5). Expression of alpha-SM actin and SM myosin heavy chain was determined using immunocytochemical staining and Western blot analysis. Baseline cell proliferation and the mitogenic response to angiotensin II was assessed by cell counting and cell viability assays. Cell contractility was determined for normal and neuropathic SMCs using an in vitro collagen lattice assay. Cell adherence was measured assessed using partial and complete trypsinization assays. RESULTS: Normal and neuropathic SMCs showed similar morphology in culture, and similar patterns of alpha-SM actin and SM myosin expression. Following 10 days of plating under optimal growth conditions the number of neuropathic SMCs was 170% more than normal SMCs. In response to angiotensin II neuropathic SMCs reached 54% of maximal growth capacity as opposed to 30% for normal SMCs (p <0.01). Neuropathic SMCs contracted significantly less in 10% serum and calcium ionophore (p <0.05), as determined by in vitro contractility assays. Neuropathic SMCs had 19% and 30% less adherent cells than normal SMCs (p <0.01) following isotonic solution washes and trypsinization, respectively. CONCLUSIONS: These results demonstrate that cultured neuropathic bladder SMCs possess and maintain different characteristics than normal SMCs in vitro. The potential clinical implications of using these cells in conjunction with tissue engineering techniques for the promotion of bladder regeneration requires further investigation.     

Construction of functional soft tissues from premodulated smooth muscle cells using a bioreactor system

Artificial smooth muscle tissues should be constructed with well-differentiated and aligned smooth muscle cells (SMCs) for proper functioning. In a previous study, we produced cell/scaffold hybrids composed of consistently aligned SMCs in a contractile state using cyclic mechanical strain. In this study, the preconditioned hybrids were organized as functional smooth muscle constructs, which had a high cellular density, using a bioreactor system. We determined that the alignment and contractile phenotype of the initially generated SMCs would be retained after a 7-day culture period in a bioreactor. Mechanical properties of the smooth muscle constructs were measured and compared with those of native smooth muscle tissues and acellular scaffolds. The constructs had a denser cell concentration than the preconditioned hybrids, although they were not fully filled with cells. The premodulated cell alignment and contractile phenotype were retained after culture in a bioreactor. The 7-day-cultured constructs had similar allowed stress levels to native tissues while their stiffness was much lower, suggesting that they had malleable and durable characteristics. These results suggest that functional smooth muscle tissues with mechanical stability can be produced using premodulated SMCs and a bioreactor system.     

Culture of vascular smooth muscle cells from small arteries of the rat kidney

BACKGROUND: In contrast to arterioles, small arteries appear to be the preferential site of renal vascular smooth muscle cell (VSMC) proliferation under pathophysiological conditions. To date, techniques have been described to isolate renal arterioles and to culture VSMCs. The aim of the present study was to develop a method of culturing VSMCs from isolated small arteries of the rat kidney and to characterize their growth as compared with that of aortic VSMCs. METHODS: Renal vascular trees were isolated from kidneys of male Wistar rats by a sieving technique. VSMCs were grown from explants of collagenase-treated renal vascular trees and thoracic aorta. Growth curves and proliferation of renal and aortic VSMCs in response to fetal bovine serum (FBS) were compared by determination of cell number and DNA synthesis, measured as incorporation of 5-bromo-2'-deoxyuridine. RESULTS: Renal vascular trees consisted mainly of small arteries with a diameter of 80 to 400 microm (interlobar and arcuate arteries). As compared with total kidney or renal cortex, alkaline phosphatase activity was decreased by 81%, and vasopressin (10 micromol/L) was unable to stimulate adenylyl cyclase in renal vascular trees, indicating little tubular contamination. A homogenous population of spindle-shaped cells was cultured from renal vascular trees, which grew in a hill-and-valley pattern and stained positively for smooth muscle alpha-actin, according to the characteristics of VSMC phenotype. Renal VSMCs proliferated more slowly than aortic VSMCs and reached the plateau of growth at about half of the cell density of aortic VSMCs. Furthermore, proliferation of renal VSMCs depended more heavily on FBS concentration, since about threefold higher concentrations of FBS were needed for renal VSMCs to multiply at the same rate and to similarly stimulate DNA synthesis as compared with aortic VSMCs. CONCLUSIONS: We present a method to culture renal VSMCs from small arteries of the rat kidney, which possess distinct growth characteristics as compared with aortic VSMCs.     

Role of Akt signaling pathway in delayed cerebral vasospasm after subarachnoid hemorrhage in rats

Background

Akt plays an important role in cell survival, proliferation, apoptosis and other activities. It also has been involved in maintaining smooth muscle cell contraction phenotypes in vitro and in vivo. Recent studies have focused on the inhibition of Akt in acute vasospasm and neuronal apoptosis after subarachnoid hemorrhage (SAH). However, its role in delayed cerebral vasospasm (DCVS) has not been reported.

Methods

In this study, using a “two-hemorrhage” rat model of SAH, we examined the expression of p-Akt and the formation of vasospasm in the basilar arteries. To investigate the possible role of Akt in phenotypic switching, we performed immunohistochemical staining to examine expressions of SMα-actin and proliferating cell nuclear antigen (PCNA), markers of smooth muscle phenotypic switching.

Results

We found that the basilar arteries exhibited vasospasm after SAH and that vasospasm became most severe on day 7 after SAH. Elevated protein expression of p-Akt was detected 4 days after SAH induction, peaked on day 7, and recovered on day 21, which was in a parallel time course to the development of DCVS. Moreover, results of immunohistochemical staining revealed enhanced expression of PCNA but gradual reduction in expression of SMα-actin from day 1 to day 7 after SAH; then, the expressions of PCNA and SMα-actin gradually recovered until day 21.

Conclusions

These results support a novel mechanism in which the Akt signaling pathway plays an important role in the proliferation of smooth muscle cells (SMCs) rather than inducing phenotype switching in basilar arteries, which promotes the development of DCVS after SAH.     

Angiogenesis inhibitor TNP-470 (AGM-1470) suppresses vascular smooth muscle cell proliferation after balloon injury in rats

BACKGROUND: Although TNP-470, a synthetic analog of fumagillin, may inhibit vascular intimal hyperplasia, the effects of TNP-470 on smooth muscle cell (SMC) proliferation have not been demonstrated in vivo. The aim of this study was to confirm the effect of TNP-470 on vascular SMC proliferation using a rat carotid artery balloon injury model. MATERIALS AND METHODS: Rats were treated with vehicle or with TNP-470 at low dosage (10 mg/kg), medium dosage (20 mg/kg), or high dosage (40 mg/kg). The animals received subcutaneous injections of materials three times a week from the day following balloon injury. All rats were sacrificed at 2 weeks after injury. The ratio of intimal-to-medial cross-sectional areas (I/M ratio) and the PCNA labeling index was calculated for each group. The DNA synthesis of cultured SMCs was also evaluated using [3H]thymidine incorporation assays. Smooth muscle cells were stimulated with basic fibroblast growth factor and TNP-470 (0.01-100 ng/ml) were added. RESULTS: The inhibition of intimal hyperplasia increased in a dose-dependent manner. TNP-470 also decreased PCNA expression in the neointima and inhibited DNA synthesis of cultured SMCs. CONCLUSIONS: TNP-470 may be useful in the prevention of vascular intimal hyperplasia.     

膀胱平滑肌细胞与小肠黏膜下层体外复合培养的实验研究

目的探讨体外快速培养犬膀胱平滑肌细胞的方法及观察膀胱平滑肌细胞在脱细胞小肠黏膜下层(small intestinal submucosa,SIS上的生长状况,为构建组织工程膀胱平滑肌组织提供实验依据。方法分别采用酶消化法和组织块培养法分离、获取和原代培养犬膀胱平滑肌细胞,倒置相差显微镜下观察细胞的生长情况,透射电镜观察细胞的超微结构,免疫组织化学染色进行细胞鉴定。将犬膀胱平滑肌细胞接种到SIS支架材料上,于复合培养5、7及9d取材,行苏木素染色、石蜡切片HE染色和扫描电镜观察膀胱平滑肌细胞在SIS上的生长状况。以细胞-SIS复合培养组为实验组,以膀胱平滑肌细胞为对照组,每组各设9孔,分别于接种后3、5及7d取材,酶消化后收集细胞并计数。结果酶消化法原代培养获取犬膀胱平滑肌细胞数量多,细胞生长速度快,形态良好,培养5d细胞在培养瓶底生长汇合。组织块培养法接种3d见长梭形的膀胱平滑肌细胞从植块边缘萌出,获取的细胞数量较少。透射电镜下见膀胱平滑肌细胞胞质中有特征性细肌丝和细胞膜的密斑。抗α-肌动蛋白免疫组织化学染色胞浆呈棕黄色阳性反应。膀胱平滑肌细胞在SIS表面能黏附、生长和增殖。体外复合培养5d后,膀胱平滑肌细胞铺满SIS表面,呈单层细胞结构。7、9d细胞形态与5d相似。实验组3、5及7d的细胞计数分别为(16.85±0.79)×105、(39.74±2.16)×105及(37.15±2.02)×105个,对照组分别为(19.43±0.54)×105、(34.50±1.85)×105及(33.07±1.31)×105个。两组5d细胞计数差异有统计学意义(P<0.05)。结论酶消化法原代培养膀胱平滑肌细胞可提供大量活性良好的种子细胞。SIS支持膀胱平滑肌细胞黏附和生长,可为构建组织工程膀胱平滑肌组织提供良好支架。     

Inhibition of smooth muscle cell proliferation and migration in vitro by antisense oligonucleotide to c-myb

Purpose: Smooth muscle cell (SMC) migration and proliferation are prominent features of intimal hyperplasia. Previous studies have shown that inhibition of c- myb inhibits arterial SMC proliferation. Our goal was to evaluate the effect of an antisense oligonucleotide targeted to c- myb on the proliferation and migration of SMC explanted from synthetic vascular grafts.Methods: SMCs were enzymatically removed from aortas and Dacron grafts explanted from dogs (n = 5). For proliferation studies, quiescent SMCs were incubated with either 0.0, 0.5, 5.0, or 10.0 μM antisense (GTGTCGGGGTCTCCGGGC) or sense (GCCCGGAGACCCCGACAC) oligonucleotides to c- myb . Proliferation was measured after 24 hours by incorporation of [ ³ H]thymidine. Migration was assessed 24 hours after a razor injury.Results: Antisense to c- myb consistently inhibited proliferation and migration of both native aortic and graft SMCs in a dose-dependent fashion. At a concentration of 10 μM antisense oligonucleotide, aortic and graft SMC proliferation rates were 32% ± 20% and 56% ± 9% of control samples, respectively. At 25 μM antisense, the number of migrating aortic and graft SMCs decreased to 41.9% ± 26.8% and 51.9% ± 34.1% of control samples, respectively.Conclusions: Our results suggest that antisense oligonucleotides to c- myb may be useful in the inhibition of SMC proliferation and migration associated with development of intimal hyperplasia. (J Vasc Surg 1996;23:783-91.)     

Different responses by cultured aortic and venous smooth muscle cells to gamma radiation

BACKGROUND: Stenosis of hemodialysis arteriovenous grafts is usually focal and caused by the proliferation of vascular smooth muscle cells (SMCs). External radiation of the graft is a potential strategy to prevent stenosis; however, the relative responsiveness of arterial and venous SMCs to radiation is unknown. METHODS: Human aortic and saphenous vein SMCs were cultured in a medium containing growth factors and serum and treated with 0 to 50 Gy in a gamma irradiator. At 2 to 20 days post-irradiation, cell counting, methylthiazoletetrazolium dye reduction, [(3)H]-thymidine uptake, and bromodeoxyuridine (BrdU) incorporation assays were performed. RESULTS: All assays showed that 1 to 50 Gy inhibited the proliferation of both aortic and venous SMCs in a dose-dependent manner. Importantly, venous cells were less susceptible to radiation in all assays, compared to aortic cells. At day 10, 1 to 50 Gy of radiation inhibited the increase in the number of aortic cells by 24% to 66% and venous cells by 8% to 25% (P < 0.01) (aortic vs. venous). The differences between aortic and venous cells varied among different assays and were most pronounced in the BrdU assay. CONCLUSION: Inasmuch as myointimal hyperplasia occurs at both arterial and venous anastomoses, future strategies using radiation to prevent hemodialysis vascular access stenosis should take these differences into consideration.     

Enhanced collagen production by smooth muscle cells during repetitive mechanical stretching

We examined the effect of repetitive mechanical stretching on smooth muscle cell (SMC) collagen production. Porcine SMCs from passages 3 through 7 were seeded in 35-mm2 flexible-bottomed culture wells at a concentration of 2 x 10(5) cells per well and allowed to attach for 24 hours. The experimental group was placed in a vacuum-operated stress-providing instrument that exerted an average elongation of 25% at maximum downward deflection of the culture plate bottom. The stretched cells (nine wells per day) were subjected to a cyclic force regimen of 10 s of elongation and 10 s of relaxation for five days. The control cells (nine wells per day) were subjected to incubation conditions similar to those in the experimental group but without cyclic stretching. Twenty-four hours before harvesting, serum-free medium containing 50-microCi tritiated proline, an amino acid hydroxylated in collagen (hydroxyproline), and 50 micrograms/mL of ascorbate was added per well. On days 3 and 5 the medium and cells were collected, precipitated with trichloroacetic acid, and then sedimented, lyophilized, and analyzed to separate hydroxyproline and proline. Values for collagen and noncollagen protein were calculated after quantitation of the hydroxyproline and proline concentrations. The results indicate that three-cycle-per-minute stretching coordinately stimulated SMC production of collagen and noncollagen protein. We conclude that pulsatile stretch enhances collagen and noncollagen protein synthesis.     

Effects of ginsenoside on large-conductance K(Ca) channels in human corporal smooth muscle cells

Ginseng was known to be an effective natural product that enhances penile erection. However, the precise biological function and mechanisms of action of ginseng with regard to erectile function remain unknown. The principal objective of this study was to identify ginsenoside (principal molecular ingredients of ginseng)-induced activation of large-conductance K(Ca) channel in human corporal smooth muscle cells, and to determine ginseng's mechanism of action on penile erection. Electrophysiological studies using cultured human corporal smooth muscle cells were conducted. We evaluated the effects of total ginsenosides (TGS) and ginsenoside Rg3 on large-conductance K(Ca) channel by determining whole-cell currents and single-channel activities. There was an increase in outward current dependent on TGS concentration (at +60 mV, 1 μg ml(-1); 168.3±59.3%, n=6, P<0.05, 10 μg ml(-1); 173.2±36.8%, n=4, P<0.05, 50 μg ml(-1); 295.3±62.3%, n=19, P<0.001, 100 μg ml(-1); and 462.3±97.1%, n=5, P<0.001) and Rg3 concentration (at +60 mV, 1 μM (0.78 μg ml(-1)); 222.8±64.8%, n=11, P<0.0001, 10 μM; 672.6±137.1%, n=10, P<0.0001, 50 μM; and 1713.3±234.7%, n=15, P<0.001) in the solution that was blocked completely by tetraethylammonium (TEA). Channel opening in cell-attached mode and channel activity in the inside-out membrane patches was also increased significantly by 50 μg of TGS or 10 μM of Rg3. The results of this study suggested that the activation of large-conductance K(Ca) channels by ginsenoside could be one mechanism of ginsenoside-induced relaxation in corporal smooth muscle.     

Heterotypic smooth muscle cell/endothelial cell interactions differ between species

BACKGROUND: In vitro coculture models have been used to study heterotypic cell-cell interactions. This study was performed to determine if species of cell origin affects heterotypic smooth muscle cell (SMC) endothelial cell (EC) interactions in coculture. METHODS: To study the effect of ECs on SMC proliferation, ECs were cultured on porous Dacron membranes. SMCs were added opposite the ECs or on bare membranes on Day 3, and after 4 days, cells were harvested for cell counts. To study the effect of SMCs on EC proliferation, ECs at a density of 5 x 10(5) cells/membrane were added to bare membranes or on membranes opposite SMCs plated 2 days earlier. After 48 h, cells were harvested for cell counts. (N = 3/condition, experiments repeated x2.) Cells of human and bovine aortic origin were used. RESULTS: The effect of coculture on cell growth differed between species. The effect of heterotypic interactions between human cocultured cells was coinhibitory on the rate of growth as compared to the growth of cells cultured alone. Growth of cocultured ECs was 55.2 +/- 8.7% less than that of ECs cultured alone while growth of cocultured SMCs was 27.2 +/- 6.0% less than growth of SMCs cultured alone. This contrasted with the bovine EC stimulation of SMC proliferation, with 66.8 +/- 5.0% greater growth of cocultured SMCs compared to SMCs cultured alone, and failure of bovine SMCs to decrease EC proliferation. CONCLUSIONS: Since significant differences in cell-material interactions occur in vivo between species, the finding that in vitro heterotypic cell-cell interactions are species dependent is not surprising. This fundamental difference in cell behavior stresses the potential importance of using human cells in studies evaluating cell-cell and cell-material interactions in vitro.     

Resveratrol inhibits vascular smooth muscle cell proliferation and induces apoptosis

OBJECTIVE: In France, despite a high intake of dietary cholesterol and saturated fat, the cardiovascular death rate is one of the lowest among developed countries. This "French paradox" has been postulated to be related to the high red wine intake in France. The aim of this study was to determine the effects of resveratrol, a major polyphenol component of red wine, on vascular smooth muscle cell (SMC) proliferation in vitro. METHODS: SMCs were exposed to 10(-6) to 10(-4) M resveratrol and cell proliferation was assessed by cell counting. Cell cycle analysis was done by treating cells with propidium iodide followed by flow-activated cell sorting. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling staining. RESULTS: We demonstrate that resveratrol inhibited bovine aortic SMC proliferation in a dose-dependent manner. The lowest concentration of resveratrol resulting in a significant decrease in SMC proliferation compared with control was 10(-5) M. By flow cytometry, we observed a block in the G1-S phase of the SMC cycle. Resveratrol treatment also resulted in a dose-dependent apoptosis of SMCs but had no effects on SMC morphology. CONCLUSION: The results indicated that vascular SMC proliferation could be inhibited by resveratrol through a block on G1-S phase and by an increase in apoptosis. It supports the conjecture that red wine consumption may have a beneficial effect on cardiovascular mortality. CLINICAL RELEVANCE: Our results suggest that resveratrol inhibits, in a dose-dependent manner, smooth muscle cell proliferation, which may help to partially explain a beneficial effect of wine drinking. This inhibition is related to an early block in the cell cycle and also to a dose-dependent apoptotic effect. The present study demonstrates that resveratrol not only is an indirect marker of a healthy life style and alimentation but may also be directly responsible for the French paradox.     

Potential differentiation of human mesenchymal stem cell transplanted in rat corpus cavernosum toward endothelial or smooth muscle cells

One of the causes of erectile dysfunction (ED) is the damaged penile cavernous smooth muscle cells (SMCs) and sinus endothelial cells (ECs). To investigate the feasibility of applying immortalized human mesenchymal stem cells (MSCs) to penile cavernous ECs or SMCs repair in the treatment of ED, the in vivo potential differentiation of the immortalized human MSCs toward penile cavernous endothelial or smooth muscle was investigated. One clone of immortalized human bone marrow mesenchymal stem cell line B10 cells via retroviral vector encoding v-myc were transplanted into the cavernosum of the Sprague-Dawley rats and harvested 2 weeks later. The expression of CD31, von Willebrand factor (vWF), smooth muscle cell actin (SMA), calponin and desmin was determined immunohistochemically in rat penile cavernosum. Multipotency of B10 to adipogenic, osteogenic or chondrogenic differentiation was found. Expression of EC specific markers (CD31 or vWF protein) and expression of SMC specific markers (calponin, SMA or desmin protein) were demonstrated in grafted B10 cells. When human MSCs were transplanted into the penile cavernosum, they have the potential to differentiate toward ECs or SMCs. Human MSCs may be a good candidate in the treatment of penile cavernosum injury.