Human Smooth Muscle Cells In Culture Research Articles

Protective Role of Postbiotic Mediators Secreted by Lactobacillus rhamnosus GG Versus Lipopolysaccharide-induced Damage in Human Colonic Smooth Muscle Cells.

Some beneficial effects of probiotics may be due to secreted probiotic-derived factors, identified as "postbiotic" mediators. The aim of this study was to evaluate whether supernatants harvested from Lactobacillus rhamnosus GG (LGG) cultures (ATCC53103 strain) protect colonic human smooth muscle cells (HSMCs) from lipopolysaccharide (LPS)-induced myogenic damage. LGG was grown in de Man, Rogosa, Share medium at 37°C and samples were collected in middle and late exponential, stationary, and overnight phases. Supernatants were recovered by centrifugation, filtered, and stored at -20°C. The primary HSMCs culture was exposed for 24 hours to purified LPS of a pathogen strain of Escherichia coli (O111:B4) (1 μg/mL) with and without supernatants. Postbiotic effects were evaluated on the basis of HSMCs morphofunctional alterations and interleukin-6 (IL-6) production. Data are expressed as mean±SE (P<0.05 significant). LPS induced persistent, significant, 20.5%±0.7% cell shortening and 34.5%±2.2% decrease in acetylcholine-induced contraction of human HSMCs. These morphofunctional alterations were paralleled to a 365.65%±203.13% increase in IL-6 production. All these effects were dose-dependently reduced by LGG supernatants. Supernatants of the middle exponential phase already partially restored LPS-induced cell shortening by 57.34%±12.7% and IL-6 increase by 145.8%±4.3% but had no effect on LPS-induced inhibition of contraction. Maximal protective effects were obtained with supernatants of the late stationary phase with LPS-induced cell shortening restored by 84.1%±4.7%, inhibition of contraction by 85.5%±6.4%, and IL-6 basal production by 92.7%±1.2%. LGG-derived products are able to protect human SMCs from LPS-induced myogenic damage. Novel insights have been provided for the possibility that LGG-derived products could reduce the risk of progression to postinfective motor disorders.

Magnetic Bacterial Nanocellulose for neurovascular reconstruction in cerebral aneurysm treatment applications

Event Abstract Back to Event Magnetic Bacterial Nanocellulose for neurovascular reconstruction in cerebral aneurysm treatment applications Sandra L Arias1, 2*, Akshath Shetty2, 3, Angana Senpan2, Monica Echeverry-Rendon4 and Jean Paul Allain2, 3 1 University of Illinois at Urbana-Champaign, Department of Bioengineering, United States 2 University of Illinois at Urbana-Champaign, Micro and Nanotechnology Laboratory, United States 3 University of Illinois at Urbana-Champaign, Department of Nuclear, Plasma and Radiological Engineering, United States 4 University of Antioquia, Department of Materials Engineering, Colombia Introduction: Cerebral aneurysms are abnormal blood pockets that form outside of the arterial wall in the brain. The main obstacle in aneurysmal neuroendovascular reconstruction approaches is the delayed closure of the aneurysm due to poor cell retention[1]. We hypothesized that a magnetic bacterial nanocellulose (MBNC) can improve local cell retention at the site of the aneurysmal neck defect via focal magnetic attractive forces. Bacterial nanocellulose (BNC) is a natural hydrogel produced by A. Xylinum, which has high mechanical properties (114 GPa for a single filament of BNC)[2], and is biocompatible[3]. To test the feasibility of our approach, we precipitated iron oxide nanoparticles (IONPs) in BNC pellicles to yield a magnetic hydrogel, and characterized the resulting material via XPS, SEM and vibrating sample magnetometer (VSM). We additionally introduced cell adhesion sites (collagen) on the MBNC via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) activation of the cellulose fibrils. A cytotoxicity test based on intracellular esterase activity was used to test the biocompatibility of the MBNC pellicles. Materials and Methods: Magnetic functionalization: The MBNC was prepared by in-situ precipitation of Fe2+ and Fe3+ ferrous ions in never-dried BNC[4]. Collagen bio-conjugation: 100 ul of CDAP solution was added on top of BNC and MBNC pellicles and allow to react for 1 min. Subsequently, 0.1 N of HCl was added per sample to stop the activation. The pellicles were washed with PBS and 200 ul of collagen was added on top per sample. The biocompatibility of the samples was tested via calcein/ethidium homodiamer-1 to mark healthy and apoptotic human smooth muscle cells (HSMCs) respectively. MBNC characterization: XPS was employed to reveal the chemical composition both of MBNC and collagen-bioconjugated MBNC pellicles. Magnetic strength via VSM was used to measure the magnetic strength of the MBNC. SEM was used to examine the morphology of the MBNC. Results and Discussion: Fig. 1A shows the set-up used in the fabrication of MBNC. The BNC acquires a black color after incorporation of IONPs, which are dispersed along the BNC fibers or forming aggregates (Fig. 1B-D). Compared with the XPS of BNC (Fig. 2), the spectrum of MBNC shows a significant new signal at a binding energy (BE) of 722 and 709 eV, which is the characteristic peak of Fe 2p1/2 and Fe 2p3/2. The XPS on collagen-conjugated MBNC resulted on the detection of nitrogen, which can be interpreted as an indirect measure of the protein content. Fig. 3 shows the esterase activity measured via calcein (green) of HSMCs culture on MBNC for 24 hours. Both samples show a high HSMC viability and confluence, indicanting that the MBNC and collagen-bioconjugated MBNC did not have negative detrimental effects on HSMCs under these experimental conditions. . The VSM plot of MBNC provide evidence that IONPS are superparamagnetic at room temperature. Conclusion: Our results show that we successfully incorporated Fe3O4 nanoparticles and cell-adhesion sites on the BNC. The VSM data showed that MBNC had a magnetic saturation of (0.054), which is above of the value considered to have satisfactory magnetic strength for tissue engineering purposes (0.049 emu/g). This work was funded by Department of Defense under contract No. W81XWH-11-2-0067

OP‐3 THE PROTECTIVE ROLE OF LACTOBACILLUS RHAMNOSUS GG‐DERIVED FACTORS AGAINST LPS‐INDUCED DAMAGE OF HUMAN COLONIC SMOOTH MUSCLE CELLS.

Impaired gut barrier function has been reported in some functional gastrointestinal (GI) disorders.Evidences suggest that gut microbiota affects GI motility in particular Lactobacillus species elicits anti‐inflammatory activity and exerts protective effects on damage induced by pathogen Gram negative‐derived lipopolysaccharide(LPS). LPS produced an oxidative imbalance in human colonic smooth muscle cells (SMC) that persists after LPS‐washout and contributes to SMC morphofunctional alterations. Aim: evaluate if supernatants harvested from LGG cultures protect SMC from LPS‐induced myogenic damage.Methods:L. rhamnosus GG (ATCC 53103 strain) was grown in MRS medium and samples were collected from bacterial cultures in middle exponential phase, in early, in middle and late stationary phase (overnight).Supernatants were recovered, filtered and stored at ‐20 °C. Highly pure human SMC culture was then exposed for 24 h to highly purified LPS (1 μg/ml) of E.coli (O111:B4) in the absence and presence of the supernatants.Their effects were evaluated on LPS‐induced SMC morphofunctional alterations and pro‐inflammatory IL‐6 production. Data are expressed as mean ± SE (p &lt; 0.05 significant).Results:LPS induced persistent significant 20.7% ± 1.2 cell shortening and 35.2% ± 2.6 decrease in contraction of human colonic SMC. These alterations were paralleled to a 238.5% ± 82.5 increase in IL‐6 production.These effects disappeared in the presence of LGG‐supernatants, following a progression related to LGG growth curve phases. Supernatants collected in the middle exponential phase already significantly partially restored LPS‐induced cell shortening by 43.4% ± 10.2 and IL6 increase by 47.6% ± 13.1 but had no effect on LPS‐induced inhibition of contraction. Supernatants collected later, in the early and middle stationary phase, further counteract LPS‐induced damage, including inhibition of contraction. Maximal protective effects were observed with supernatants of the late stationary phase where LPS‐induced cell shortening was reversed by 86% ± 4.7, inhibition of contraction by 98.2% ± 1.8 and IL6 basal production by 91.3% ± 0.6.Conclusions: LGG secreted products are substances/byproducts able to directly protect human SMC from LPS‐induced myogenic damage. Novel insights are then provided about the possibility that LGG‐derived products could reduce the risk of progression to a post‐infective motor disorder.

C-reactive protein induces expression of matrix metalloproteinase-9: A possible link between inflammation and plaque rupture

Matrix metalloproteases (MMPs) have been implicated in the pathogenesis of acute coronary syndromes (ACS). However, little is known about the mechanisms responsible for MMP expression in ACS. C-reactive protein (CRP) not only is an independent risk factor for cardiovascular events, but also may exert direct pro-atherosclerotic effects. Therefore, we aimed at determining whether CRP might induce MMP-9 in two different experimental conditions: 1) smooth muscle cells (SMCs) in vitro, and 2) patients with ACS. Effects of increasing concentrations of CRP on MMP-9 expression were evaluated in vitro in human SMCs. TIMP-1 protein expression, the selective inhibitor of MMP-9, was also evaluated. CRP dose-dependently induced MMP-9 expression in SMCs by promoting MMP-mRNA transcription, as well as MMP-9 secretion. In contrast, no differences were found for TIMP-1 protein expression. In vivo, MMP-9 and CRP levels were measured in blood samples obtained from the aorta (Ao) and the coronary sinus (Cs) of patients with normal coronary arteries (controls, n=21), stable angina (n=24), and ACS (n=30). Both MMP-9 and CRP plasma levels were significantly increased across the coronary circulation only in patients with ACS. Interestingly, a significant correlation between MMP-9 and CRP plasma levels was found. CRP induced MMP-9 expression and activity in human SMCs in culture; patients presenting with ACS have increased transcoronary plasma levels of MMP-9 and CRP with a significant correlation between these two markers. This may explain the heightened risk of coronary events in subjects with elevated levels of CRP.

Modified explant technique for the culture of human vascular smooth muscle cells

Objective To establish an effective method of human vascular smooth muscle cells in vitro culture.Methods Medial layer of human aorta was separated under sterile conditions,then cut into small pieces (about 1 mm3).After pretreatment with 0.1％ type Ⅰ collagenase,the tissues were transferred into flask and cultured in DMEM containing 20％ fetal bovine serum,penicillin (100 U/ml) and streptomycin (100 g/L).The cell morphology was observed under the inverted microscopy.Smooth muscle cell specific protein (α-SMA) was identified by using immumofluorescence methods.Results Some smooth muscle cells started growing from explants in 4 to 5 days.One to 2 weeks later,the cells grew to confluence by migrated cells.Immumofluorescence staining showed positive expression of α-SMA (fluorescence intensity (++)-(+++)).Conclusion Modified explant technique by pretreating tissues with collagenase was an effective method to get vascular smooth muscle cells in vitro. Key words: Aorta; Vascular smooth muscle cells; Cell culture

Hepatocyte Growth Factor/Scatter Factor and MET Are Involved in Arterial Repair and Atherogenesis

Several studies have shown that in the arterial wall hepatocyte growth factor/scatter factor (HGF/SF) is expressed by smooth muscle cells (SMCs) but acts on endothelial cells, not SMCs. Other studies, however, have indicated that SMCs can respond to HGF/SF. We have reinvestigated expression and activity of HGF/SF and its receptor MET in arterial SMC and endothelial cell cultures and in whole arteries after superficial or deep injury or atherogenesis. High-density cultures of SMCs produced HGF/SF but did not express MET, whereas SMCs, at the leading edge of injured cultures, expressed both ligand and receptor and showed a dramatic motility and growth response to HGF/SF. In line with these results, HGF/SF and MET expression was undetectable in the media of uninjured carotid arteries but was induced after deep arterial injury in areas of SMC migration in the neointima. Strong MET expression was also observed in the SMCs of the atherosclerotic lesions of homozygous apoE(-/-) mice, whereas HGF/SF was expressed by macrophage-derived foam cells. These results demonstrate that MET is induced in migrating and proliferating SMCs and that HGF/SF and MET are key mediators of the SMC response in atherogenesis.

Elevated extracellular calcium levels induce smooth muscle cell matrix mineralization in vitro

Hyperphosphatemia, elevated calcium x phosphorus product (Ca x P), and calcium burden, major causes of vascular calcification, are correlated with increased cardiovascular morbidity and mortality in dialysis patients. To address the underlying mechanisms responsible for these findings, we have utilized an in vitro human smooth muscle cell (HSMC) model of vascular calcification. Previous studies using this system demonstrated enhanced calcification of HSMC cultures treated with phosphorus levels in the hyperphosphatemic range, and implicated a sodium-dependent phosphate cotransport-dependent mechanism in this effect. In the present study, we examine the effect of increasing calcium concentrations on HSMC calcification in vitro. Increasing calcium to levels observed in hypercalcemic individuals increased mineralization of HSMC cultures under normal phosphorus conditions. Importantly, at these total calcium concentrations, ionized calcium levels increased from 1.2 mmol/L to 1.7 mmol/L, consistent with levels observed physiologically in normocalcemic and hypercalcemic individuals, respectively. Furthermore, increasing both calcium and phosphorus levels led to accelerated and increased mineralization in the cultures. Calcium-induced mineralization was dependent on the function of a sodium-dependent phosphate cotransporter, since it was inhibited by phosphonoformic acid (PFA). While elevated calcium did not affect short-term phosphorus transport kinetics, long-term elevated calcium treatment of HSMCs induced expression of the sodium-dependent phosphate cotransporter, Pit-1. These studies suggest that elevated calcium may stimulate HSMC mineralization by elevating Ca x P product and enhancing the sodium-dependent phosphate cotransporter-dependent mineralization pathway previously observed in HSMCs.

Rapamycin reduces neointima formation during vascular injury.

Proliferation and migration of vascular smooth muscle cells (SMCs) mark the key processes in the development of bypass graft disease and during neointima formation in restenosis after angioplasty. Growth factors are potent SMC mitogens as they are involved in SMC proliferation and in extracellular matrix (ECM) synthesis. Based on these premises, we examined the effect of the proliferation inhibitor rapamycin in human SMC culture and in a rabbit vascular injury model. Injection of rapamycin or its vehicle was performed with an infusion-balloon catheter directly into the vessel wall during vascular injury. The intima/media ratio was determined histologically whereas the protein expression was analysed using the powerful two-dimensional gel electrophoresis (2D page) technique. Inhibition of proliferation after rapamycin application was estimated in a human SMC culture for time and dose dependent effects. Rapamycin treatment resulted in a significant reduction of intima media ratio compared to vehicle treated animals after three weeks (0.65 +/- 0.1 vs. 1.2 +/- 0.2 intima-media-ratio, p < 0.05). 2D electrophoresis analysis proved increased ECM synthesis following angioplasty (i.e., lamin, vimentin) in vehicle treated animals. Local rapamycin administration resulted in profound reduction of ECM synthesis after vascular injury. In in-vitro experiments exposure of cultured human SMCs to rapamycin resulted in a significant and dose-dependent (1 nm-100 nm) reduction of human smooth muscle cell proliferation measured by cell counting. These above mentioned results suggest that protein synthesis in addition to reduction of cellular proliferation plays an important role following vascular injury, since application of rapamycin resulted in the reduction of SMC proliferation and ECM-synthesis.

α5β1 Integrin Expression and Luminal Edge Fibronectin Matrix Assembly by Smooth Muscle Cells after Arterial Injury

Fibronectin is secreted from the cell as a soluble protein that must then polymerize to regulate cell function. To elucidate the process of fibronectin matrix assembly in vascular disease, we immunostained sections of balloon-injured rat carotid artery for the fibronectin-binding alpha5beta1 integrin. Whereas alpha5beta1 integrin was not evident in the normal carotid artery, its expression was induced after a vascular injury. By 14 days, the alpha5beta1 integrin was localized exclusively to the less differentiated smooth muscle cells (SMCs) at the luminal surface of the neointima. Platelet-derived growth factor-BB, dominant in neointimal formation, selectively increased the expression of the alpha5beta1 integrin by human SMCs in culture. To track the assembly of fibronectin fibers, fluorescence-labeled soluble fibronectin protomers were added to cultured SMCs and to fresh segments of normal and balloon-injured rat carotid arteries. Fibronectin fiber formation in cultured SMCs could be detected within 10 minutes, and was blocked by an RGD peptide, an anti-beta1 integrin antibody, and an anti-alpha5beta1 integrin antibody, but not by an anti-beta3 integrin antibody. En face confocal microscopy of arterial segments revealed that soluble fibronectin had polymerized on the alpha5beta1 integrin-expressing SMCs of the luminal surface of the injured arterial neointima, but not on the alpha5beta1 integrin-negative neointimal SMCs below this or on the endothelial cells of uninjured arteries. Furthermore, in situ fibronectin assembly by the neointimal SMCs was inhibited by an RGD peptide and by an anti-beta1 integrin antibody. These studies indicate that a subpopulation of SMCs in the repairing artery wall orchestrates integrin-mediated fibronectin assembly.

Potentiated angiogenic effect of scatter factor/hepatocyte growth factor via induction of vascular endothelial growth factor: the case for paracrine amplification of angiogenesis.

Scatter factor/hepatocyte growth factor (SF/HGF) is a pleiotropic growth factor that stimulates proliferation and migration of endothelial cells (ECs) via the c-Met receptor, present on ECs as well as other cell types, including smooth muscle cells (SMCs). We studied the effects of recombinant human (rh) SF/HGF in vitro and in vivo in a rabbit model of hindlimb ischemia. We further compared these effects with those of recombinant human vascular endothelial growth factor (rhVEGF165), an EC-specific mitogen. In vitro, rhSF/HGF and rhVEGF165 exhibited similar effects on proliferation and migration of ECs. When both cytokines were administered together, the result was an additive effect on EC proliferation and a synergistic effect on EC migration. Application of rhSF/HGF to cultures of human SMCs resulted in the induction of VEGF mRNA and protein. In vivo, administration of rhSF/HGF (500 microg x 3) was associated with significant improvements in collateral formation (P<.001) and regional blood flow (P<.0005) and with a significant reduction in muscle atrophy (P<.0001). These effects were significantly more pronounced than those of rhVEGF165 administered according to the same protocol (P<.05). Neither remote angiogenesis nor other pathological sequelae were observed with either rhSF/HGF or rhVEGF165. The pleiotropic effects of certain growth factors may potentiate angiogenesis via a combination of direct effects on EC proliferation and migration and indirect effects that result in the generation of other potent EC mitogens from non-EC populations. The synergistic effects demonstrated when SF/HGF and VEGF are administered together in vitro may be reproduced in vivo by SF/HGF-induced upregulation of VEGF in vascular SMCs.

Urokinase and tissue-type plasminogen activator stimulate human vascular smooth muscle cell migration

The objective of this study was to investigate the role of the plasminogen activation system in the migration of human vascular smooth muscle cells in vitro. After wounding of confluent human smooth muscle cell cultures by stripping cells from their extracellular matrix, cells start to migrate from the wounded edge into the denuded area. Addition of plasmin to the culture medium resulted in an approximately 50% increase of migrated cells after 24 hours. The plasmin inhibitor aprotinin was able to reduce this effect to control levels. Migration could also be stimulated by addition of urokinase-type plasminogen activator (HMW-u-PA) (30%) or tissue-type plasminogen activator (t-PA) (28%). Simultaneous addition of aprotinin reduced this increase below control levels, indicating that both HMW-u-PA and t-PA mediated plasminogen activation contributes to smooth muscle cell migration. Addition of low molecular weight u-PA (LMW-u-PA), a u-PA form lacking the receptor binding domain, or the aminoterminal fragment of u-PA (ATP), lacking the active site, had no effect on migration. These results suggest that both t-PA and u-PA can contribute to human smooth muscle cell migration in vitro, most likely via plasminogen activation. For the stimulation of migration by u-PA, activity as well as binding to its cell-surface receptor appears to be involved.

The effect of the neurolytic agent ethanol on cytoplasmic calcium in arterial smooth muscle and endothelium.

Celiac plexus neurolysis, although effective in relieving pain associated with upper abdominal malignancy, occasionally results in paraplegia. Diffusion of the neurolytic agent to arteries supplying the spinal cord has been postulated as a cause, and previous studies with isolated lumbar segmental arteries have demonstrated contraction in response to ethanol and phenol. The mechanism of this contractile effect is unknown, but a role for insular free calcium (Ca2+i) is suggested by the known involvement of Ca2+i in both smooth muscle vasoconstriction and toxic cell injury. The authors sought to determine whether nontoxic concentrations of ethanol cause a direct elevation of Ca2+i in arterial smooth muscle and endothelium. Primary cultures of human aortic smooth muscle and endothelial cells were studied to determine the direct effect of ethanol independent of interactions with agonists or contractile proteins. Ca2+i levels were determined in single cells with digitized video fluorescence microscopy, using ratio imaging of the Ca2+i-sensitive fluorophore fura-2. In aortic smooth muscle cells, initial Ca2+i was 98 +/- 41 nM (n = 59 cells). Histamine (10 microM) as a positive control caused an increase in Ca2+i, as expected. Ethanol alone, at doses of 2-5% (v/v) also caused a sustained elevation in Ca2+i of physiologically significant magnitude. Ethanol at doses of 5% or lower did not cause any visibly apparent injury within 30 minutes. In contrast, 10% or higher ethanol doses quickly caused membrane blebbing, a sign of toxic injury, followed by cell death within 20 minutes. Aortic endothelial cells were more resistant to ethanol than smooth muscle cells, in terms of both Ca2+i elevation and cell death. Ethanol, even at nontoxic concentrations, has a direct effect on aortic smooth muscle Ca2+i, large enough to be associated with significant vasoconstriction. The findings suggest a possible role for pharmacologic agents that preserve Ca2+i homeostasis in protecting against neurolysis-induced paraplegia, although additional study is required before clinical application is appropriate.

Unfractionated heparin and low molecular weight heparin do not inhibit the growth of proliferating human arterial smooth muscle cells in culture

To clarify the effects of unfractionated heparin (UH) and low molecular weight heparin (LMWH) on proliferating human smooth muscle cells (SMC) compared to growth arrested SMC. A cell culture study where proliferating SMC were exposed to different concentrations of UH and LMWH and the effect on proliferation and collagen secretion was studied. Growth arrested SMC were stimulated with serum and the effect of UH on proliferation was measured. Sections of Medical Angiology and Vascular Surgery, Malmö General Hospital, Sweden. Human SMC were established from arterial tissue obtained at vascular surgery or at organ donation. Effects of UH and LMWH on total cellular DNA, 3H-thymidine incorporation and collagen secretion using proliferating and growth arrested human SMC in culture. In proliferating SMC that had not been growth arrested, 1 and 10 IU/ml UH and LMWH significantly increased total cellular DNA compared to controls while DNA synthesis was not influenced. The higher cellular DNA was probably not a consequence of increased proliferation as DNA synthesis was not affected by UH or LMWH. The increased total cellular DNA could instead be due to reduced cell death. Higher concentrations (10 IU/ml) of UH and LMWH also increased collagen secretion. In control experiments with UH DNA, synthesis was decreased in stimulated human SMC that had been growth arrested previously to heparin exposure. The effects of UH and LMWH on SMC proliferation will depend on the proliferative state of the SMC. The results might be of relevance for the understanding of the atherosclerotic process and for pharmacologic interventions to prevent restenosis after angioplasty or surgery.

Low-dose, beta-particle emission from 'stent' wire results in complete, localized inhibition of smooth muscle cell proliferation.

Restenosis after catheter-based revascularization has been demonstrated to be primarily caused by medial and/or intimal smooth muscle cell (SMC) proliferation. The objective of this study was investigate the ability of local emission of beta-particles from a 32P-impregnated titanium "stent" wire source to inhibit vascular SMC and endothelial cell proliferation in cell culture and to determine the dose-response characteristics of this inhibition. A series of experiments were performed using 0.20-mm-diameter titanium wires that were impregnated with varying low concentrations of 32P (activity range, 0.002 to 0.06 microCi/cm wire, n = 47) or 31P (nonradioactive control, n = 28) in cultures of rat and human aortic SMCs and in cultured bovine aortic endothelial cells. The zone of complete cell growth inhibition (in millimeters from stent wire) was measured using light microscopy in the cultures exposed to the radioactive (32P) or control (31P) wires at 6 and 12 days after plating. In both rat and human SMC cultures there was a distinct 5.5- to 10.6-mm zone of complete SMC inhibition at wire activity levels > or = 0.006 microCi/cm. In contrast, there was no zone of inhibition surrounding the control (31P impregnated) wires (P < .001 versus 32P wires at all wire activities > or = 0.006 microCi/cm for human and rat SMCs). Proliferating bovine endothelial cells were more radioresistant than SMCs, with no zone of inhibition observed at wire activity levels up to 0.019 microCi/cm (P < .001 versus SMCs at 0.006 microCi/cm and 0.019 microCi/cm). We conclude that very low doses of beta-particle emission from a 32P-impregnated stent wire (activity levels as low as 0.006 microCi/cm of wire) completely inhibit the growth and migration of both rat and human SMCs within a range of 5.5 to 10.6 mm from the wire. Endothelial cells appear to be much more radioresistant than SMCs. These data suggest that an intra-arterial stent impregnated with a low concentration of 32P may have a salutary effect on the restenosis process. Whether this approach can be used successfully and safely to inhibit restenosis in vivo and in the clinical setting is under investigation.

Biochemical analysis of a human humoral fibroblast inhibitory factor associated with impaired vascular prosthetic graft incorporation

A specific inhibitory factor was isolated from human serum of a patient who manifested impaired prosthetic graft tissue incorporation. The later reaction was hypothesized to be related to inhibiting fibroblast proliferation by a specific humoral factor. The crude inhibitory serum was tested against a pool of normal human sera with different cell types in culture. Proliferation of human skin fibroblast and human smooth muscle cell cultures incubated in the presence of 50% inhibitory serum were inhibited up to 58% and 37%, respectively. Proliferation of bovine capillary endothelial cell cultures was stimulated under similar conditions. Isolation and purification of the inhibitory factor from crude serum were initiated by ammonium sulfate precipitation. The pellet was further fractionated by sepharose 6B gel filtration. The inhibitory activity was eluted from the column in a relatively purified form as indicated by gel electrophoresis of the inhibitory fractions, which demonstrated a specific band corresponding to the inhibitory protein, with an apparent relative molecular mass of 230 kDa. The inhibitory factor showed a high affinity to concanavalin A, indicating its nature as a glycoprotein not associated with albumin or immunoglobulin fractions of the serum.

Observations on human smooth muscle cell cultures from hyperplastic lesions of prosthetic bypass grafts: Production of a platelet-derived growth factor—like mitogen and expression of a gene for a platelet-derived growth factor receptor—A preliminary study

Prosthetic bypass grafts placed to the distal lower extremity often fail because of an occlusive tissue response in the perianastomotic region. The origin of the cells that comprise this occlusive lesion and the causes of the cellular proliferation are not known. To increase our understanding of this process we cultured cells from hyperplastic lesions obtained from patients at the time of reexploration for lower extremity graft failure, and we studied their identity and growth factor production in tissue culture. These cultures contain cells that express muscle-specific actin isoforms, shown by immunohistochemical staining, consistent with vascular smooth muscle origin. These cultures also released material that stimulated smooth muscle cell growth. A portion of this activity was similar to platelet-derived growth factor, since preincubation with antibody-to-human platelet-derived growth factor partially blocked the mitogenic effect of medium conditioned by human anastomotic hyperplastic cells. These conditioned media also contained material that competed with platelet-derived growth factor for its receptor, as measured in a radioreceptor assay. Northern blot analysis showed that these cells contain messenger RNA that encodes the A chain but not the B chain of platelet-derived growth factor. In addition, these cells contain messenger RNA that encodes a platelet-derived growth factor receptor. We conclude that cultured smooth muscle cells from human anastomotic hyperplastic lesions express genes for platelet-derived growth factor A chain and a platelet-derived growth factor receptor and secrete biologically active molecules similar to platelet-derived growth factor. These data suggest that proliferating cells adjacent to failing arterial prostheses may stimulate their own proliferation by secreting and responding to platelet-derived growth factor—like mitogens, an autostimulatory or autocrine growth effect. The elaboration of smooth muscle mitogens by cells near the anastomotic sites of small-caliber prosthetic arterial bypass grafts may contribute to the failure that limits their clinical utility.

Observations on human smooth muscle cell cultures from hyperplastic lesions of prosthetic bypass grafts: Production of a platelet-derived growth factor[mdash ]like mitogen and expression of a gene for a platelet-derived growth factor receptor[mdash ]A preliminary study

Prosthetic bypass grafts placed to the distal lower extremity often fail because of an occlusive tissue response in the perianastomotic region. The origin of the cells that comprise this occlusive lesion and the causes of the cellular proliferation are not known. To increase our understanding of this process we cultured cells from hyperplastic lesions obtained from patients at the time of reexploration for lower extremity graft failure, and we studied their identity and growth factor production in tissue culture. These cultures contain cells that express muscle-specific actin isoforms, shown by immunohistochemical staining, consistent with vascular smooth muscle origin. These cultures also released material that stimulated smooth muscle cell growth. A portion of this activity was similar to platelet-derived growth factor, since preincubation with antibody-to-human platelet-derived growth factor partially blocked the mitogenic effect of medium conditioned by human anastomotic hyperplastic cells. These conditioned media also contained material that competed with platelet-derived growth factor for its receptor, as measured in a radioreceptor assay. Northern blot analysis showed that these cells contain messenger RNA that encodes the A chain but not the B chain of platelet-derived growth factor. In addition, these cells contain messenger RNA that encodes a platelet-derived growth factor receptor. We conclude that cultured smooth muscle cells from human anastomotic hyperplastic lesions express genes for platelet-derived growth factor A chain and a platelet-derived growth factor receptor and secrete biologically active molecules similar to platelet-derived growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)