Regulation Of Smooth Muscle Cells Research Articles

660 ELECTROSPUN-ACELLULAR MATRIX COMPOSITE SCAFFOLD FOR BLADDER RECONSTRUCTION

INTRODUCTION AND OBJECTIVES: Tissue engineering aims to provide alternatives to bladder reconstruction to overcome the significant limitations associated with enterocystoplasty such as metabolic disturbance, urolithiasis, infections and even malignant diseases. As the scaffold is an important determining factor for the success of tissue engineering we have developed a composite scaffold mimicking morphological and functional characteristics of the bladder wall. A two layered construct, consistent of bladder acellular matrix (BAM) and electrospun poly(lactide-co-glycolide) (PLGA) microfibers seeded with autologous smooth muscle cells (SMCs) was developed and evaluated in a cystoplasty model in rats. METHODS: The composite scaffold was manufactured by direct electrospinning of randomly oriented PLGA fibers onto bladder acellular matrix. To assess the capability to support cell attachment, proliferation and migration autologous bladder SMCs were cultured, expanded and seeded on the polymer fibers of the composite scaffold. Electron microscopy, histological examinations and biochemical assays for cell proliferation (MTT assay) were performed. In 16 Lewis rats, partial cystectomy ( 50%) was performed, followed by augmentation cystoplasty with seeded or unseeded (control) scaffolds. Histological studies were performed after 4 and 8 weeks. RESULTS: Direct electrospinning of PLGA microfibers onto bladder acellular matrix resulted in composite scaffold with randomly oriented microstructures and remained stable in aqueous medium over prolonged time period. SEM analysis showed single fiber diameter ranging from 4–5 im. Composite scaffolds provided good support for cell attachment, proliferation and migration, and eventually developed into a bladder wall-like structure in vivo. Histological evaluation of the implanted biomaterial revealed a normally structured urothelial lining, a collagen rich compartment with early revascularisation, early smooth muscle cell infiltration and regeneration of the lamina muscularis mucosae. Immuno-histochemical analyses confirmed the urothelial and muscle cell phenotype and showed the presence of blood vessels and smooth muscle cells. CONCLUSIONS: We were able to demonstrate that a biomaterial system consisting of two completely different materials is capable to promote cell growth and proliferation in vitro and generation of bladder wall structures in vivo. This scaffold may be useful in patients requiring bladder reconstruction.

MiRNAs: roles and clinical applications in vascular disease

miRNAs are small, endogenously expressed noncoding RNAs that regulate gene expression, mainly at the post-transcriptional level, via degradation or translational inhibition of their target mRNAs. Functionally, an individual miRNA can regulate the expression of multiple target genes. The study of miRNAs is rapidly growing and recent studies have revealed a significant role of miRNAs in vascular biology and disease. Many miRNAs are highly expressed in the vasculature, and their expression is dysregulated in diseased vessels. Several miRNAs have been found to be critical modulators of vascular pathologies, such as atherosclerosis, lipoprotein metabolism, inflammation, arterial remodeling, angiogenesis, smooth muscle cell regeneration, hypertension, apoptosis, neointimal hyperplasia and signal transduction pathways. Thus, miRNAs may serve as novel biomarkers and/or therapeutic targets for vascular disease. This article summarizes the current studies related to the disease correlations and functional roles of miRNAs in the vascular system and discusses the potential applications of miRNAs in vascular disease.

Bladder tissue engineering: Tissue regeneration and neovascularization of HA‐VEGF‐incorporated bladder acellular constructs in mouse and porcine animal models

Successful tissue engineering requires appropriate recellularization and vascularization. Herein, we assessed the regenerative and angiogenic effects of porcine bladder acellular matrix (ACM) incorporated with hyaluronic acid (HA) and vascular endothelial growth factor (VEGF) in mouse and porcine models. Prepared HA-ACMs were rehydrated in different concentrations of VEGF (1, 2, 3, 10, and 50 ng/g ACM). Grafts were implanted in mice peritoneum in situ for 1 week. Angiogenesis was quantified with CD31 and Factor VIII immunostaining using Simple PCI. Selected optimal VEGF concentration that induced maximum vascularization was then used in porcine bladder augmentation model. Implants were left in for 4 and 10 weeks. Three groups of six pigs each were implanted with ACM alone, HA-ACM, and HA-VEGF-ACM. Histological, immunohistochemical (Uroplakin III, alpha-SMA, Factor VIII), and immunofluorescence (CD31) analysis were performed to assess graft regenerative capacity and angiogenesis. In mouse model, statistically significant increase in microvascular density was demonstrated in the 2 ng/g ACM group. When this concentration was used in porcine model, recellularization increased significantly from weeks 4 to 10 in HA-VEGF-ACM, with progressive decrease in fibrosis. Significantly increased vascularization, coupled with increased urothelium and smooth muscle cell (SMC) regeneration, was observed in HA-VEGF grafts at week 10 in the center and periphery, compared with week 4. HA-VEGF grafts displayed highest in vivo epithelialization, neovascularization, and SMCs regeneration. A total of 2 ng/g tissue VEGF when incorporated with HA proved effective in stimulating robust graft recellularization and vascularization, coordinated with increased urothelial bladder development and SMC augmentation into bundles by week 10.

Hypoxia regulates resistin in vascular smooth muscle cells, what next?

Hypoxia regulates resistin in vascular smooth muscle cells, what next?

Small Intestinal Submucosa as a Bioscaffold for Tissue Regeneration in Defects of the Colonic Wall

BackgroundSmall intestinal submucosa (SIS) has proved considerable regenerative capacity for repair of bowel wall defects at different locations. This study assesses the effectiveness of SIS in the repair of defects at a gastrointestinal location with strong bacterial contamination. MethodsFourteen domestic pigs had a 4.5 × 1.5 cm full-thickness defect created on the wall of the descending colon. Repair was done by suturing an SIS patch to the defect. Grafts were harvested after 30, 60, and 90 days. Outcomes were evaluated on the basis of animal survival, clinical course, and macroscopic, histological, and immunohistochemical assessment. ResultsAll animals survived the scheduled observation period. No patch failure and no postoperative leakage occurred. No luminal narrowing occurred at SIS-patched colon. Morphometric examination revealed contraction of the patched area of 77% after 30 days and more than 90% after 60 and 90 days. By 60 and 90 days, all animals showed mucosal regeneration at the margins of the graft. By 90 days, regeneration of smooth muscle cells was present at the original site of the muscularis mucosae. None of the reconstructed areas showed complete mucosal coverage or regeneration of a structured muscular layer. ConclusionSIS can be used effectively for patch repair of colonic defects in a porcine model. Distinctive contraction of the reconstructed area and limited architectural regeneration of the bowel wall suggest limitation of morphologic regenerative capacities in large-bowel regeneration.

Tubularized urethral replacement with unseeded matrices: what is the maximum distance for normal tissue regeneration?

Complete urethral replacement using unseeded matrices has been proposed as a possible therapy in cases of congenital or acquired anomalies producing significant defects. Tissue regeneration involves fibrin deposition, re-epithelialization, and remodeling that are limited by the size of the defect. Scar formation occurs because of an inability of native cells to regenerate over the defect before fibrosis takes place. We investigated the maximum potential distance of normal native tissue regeneration over a range of distances using acellular matrices for tubular grafts as an experimental model. Tubularized urethroplasties were performed in 12 male rabbits using acellular matrices of bladder submucosa at varying lengths (0.5, 1, 2, and 3 cm). Serial urethrography was performed at 1, 3, and 4 weeks. Animals were sacrificed at 1, 3, and 4 weeks and the grafts harvested. Urothelial and smooth muscle cell regeneration was documented histologically with H&E and Masson's trichrome stains. Urethrograms demonstrated normal urethral calibers in the 0.5 cm group at all time points. The evolution of a stricture was demonstrated in the 1, 2, and 3 cm grafts by 4 weeks. Histologically all grafts demonstrated ingrowth of urothelial cells from the anastomotic sites at 1 week. By 4 weeks, the 0.5 cm grafts had a normal transitional layer of epithelium surrounded by a layer of muscle within the wall of the urethral lumen. The 1, 2, and 3 cm grafts showed ingrowth and normal cellular regeneration only at the anastomotic edges with increased collagen deposition and fibrosis toward the center by 2 weeks, and dense fibrin deposition throughout the grafts by 4 weeks. The maximum defect distance suitable for normal tissue formation using acellular grafts that rely on the native cells for tissue regeneration appears to be 0.5 cm. The indications for the use of acellular matrices in tubularized grafts may therefore be limited by the size of the defect to be repaired.

Correlation of sequential nuclear imaging with interval histological changes in a porcine abdominal aortic aneurysm model

The mechanism of abdominal aortic aneurysm (AAA) formation remains incompletely described. AAA were created in the infrarenal aortas of 14 Yorkshire swine (25–35kg) using balloon dilation and luminal collagenase (8000u) and elastase (500u) perfusion. Positron emission tomography computed tomography (PET CT) was performed on post-operative days 1, 3, 7, 14, and 28 in 5 swine. Cellular uptake of 18-fluorodeoxyglucose (FDG) was quantified within AAA and sham segments of aorta using radioactive decay density within an ellipsoid volume of interest (VOI). Nine swine were euthanized at 1(n=3), 3(n=1), 7(n=1), 14(n=1), 21(n=2) and 28(n=1) days for histological analysis (H&E, Masson’s trichrome, and Verhoeff-van Giesen stain). AAA activity on day 7 (32.7%±22.3%) was significantly higher (p=0.01) than day one (−2.8±17.9%) and day 28 (1.2±22.9%). Inflammatory response, smooth muscle cell regeneration, and the appearance of hyaline matrix deposition correlate to interval FDG activity of the AAA. Extensive elastin fiber disruption was evident at all time points. FDG uptake demonstrates increased local metabolic demand following mechanical and enzyme-induced AAA formation. Regression of relative FDG uptake at 28 days suggests that elastase induced injury may not be perpetual. PET CT may provide a useful method for longitudinal noninvasive measurements within a single animal for endovascular research. Change in Relative Activity Within Ellipsoid VOIs Enclosing Porcine AAA and Sham Aorta

Amniotic Fluid and Bone Marrow Derived Mesenchymal Stem Cells Can be Converted to Smooth Muscle Cells in the Cryo-Injured Rat Bladder and Prevent Compensatory Hypertrophy of Surviving Smooth Muscle Cells

Wound healing of the cryo-injured bladder can bring about organ remodeling because of incomplete reconstitution of depleted smooth muscle cells. Stem cell transplantation could be beneficial to improve smooth muscle cell regeneration and/or modulate the remodeling process. The repair of bladder injury using adult-type stem cells would be useful for adult urological patients but unsuited for neonatal patients, in whom major benefits are likely to derive from fetal-type stem cells. The smooth muscle cell differentiation potential of fetal-type vs adult-type stem cells was evaluated by injecting green fluorescent protein labeled mesenchymal stem cells from rat amniotic fluid or bone marrow, respectively, in cryo-injured rat bladder walls. At 30 days after transplantation only a few fetal-type or adult-type mesenchymal stem cells gave rise to enteric or vascular smooth muscle cells, whereas most mesenchymal stem cells appeared incapable of specific differentiation. In vitro co-culture experiments of smooth muscle cells with fetal-type or adult-type mesenchymal stem cells selectively labeled with distinct fluorochromes showed the presence of hybrid cells, suggesting that some mesenchymal stem cells can undergo cell fusion. Surprisingly the major effect of rat bone marrow or amniotic fluid mesenchymal stem cell transplantation seemed to be preventing cryo-injury induced hypertrophy of surviving smooth muscle cells. In this model stem cell transplantation has a limited effect on smooth muscle cell regeneration. Instead it can regulate post-injury bladder remodeling, possibly via a paracrine mechanism.

Induction of Smooth Muscle Cell-Like Phenotype in Marrow-Derived Cells among Regenerating Urinary Bladder Smooth Muscle Cells

Tissue regeneration on acellular matrix grafts has great potential for therapeutic organ reconstruction. However, hollow organs such as the bladder require smooth muscle cell regeneration, the mechanisms of which are not well defined. We investigated the mechanisms by which bone marrow cells participate in smooth muscle formation during urinary bladder regeneration, using in vivo and in vitro model systems. In vivo bone marrow cells expressing green fluorescent protein were transplanted into lethally irradiated rats. Eight weeks following transplantation, bladder domes of the rats were replaced with bladder acellular matrix grafts. Two weeks after operation transplanted marrow cells repopulated the graft, as evidenced by detection of fluorescent staining. By 12 weeks they reconstituted the smooth muscle layer, with native smooth muscle cells (SMC) infiltrating the graft. In vitro, the differential effects of distinct growth factor environments created by either bladder urothelial cells or bladder SMC on phenotypic changes of marrow cells were examined. First, supernatants of cultured bladder cells were used as conditioned media for marrow cells. Second, these conditions were reconstituted with exogenous growth factors. In each case, a growth factor milieu characteristic of SMC induced an SMC-like phenotype in marrow cells, whereas that of urothelial cells failed. These findings suggest that marrow cells differentiate into smooth muscle on acellular matrix grafts in response to the environment created by SMC.

Urethral replacement: a comparison between small intestinal submucosa grafts and spontaneous regeneration

To evaluate the use of commercially available single-layer small intestinal submucosa (SIS) for urethral replacement, both as an onlay and as a tube, in a rabbit model. Thirty-six male rabbits were assigned to four experimental groups. Group 1 had the ventral wall of the penile urethra excised for 15 mm; in group 2 this created defect was patched with a SIS onlay graft; group 3 had complete excision of a 15 mm segment of the penile urethra; and in group 4, this created defect was replaced with a SIS tube graft. In all rabbits the urethra was stented for 2 weeks. A retrograde urethrogram was taken in all rabbits before death at 3, 6 and 12 weeks after surgery. The urethra was then exposed, examined carefully and excised for histopathological examination. In groups 1 and 2 the retrograde urethrograms were normal in 13 rabbits and there was relative narrowing in two rabbits in group 1 and three in group 2. In groups 3 and 4 all rabbits developed urethral fistulae or strictures. Histological examination of the urethra showed epithelial regeneration supported by smooth muscle backing in all rabbits in group 1, while rabbits in group 2 showed no regeneration of smooth muscle. By contrast, rabbits in groups 3 and 4 showed incomplete regeneration and progressive fibrosis. Single-layer SIS is not a suitable urethral substitute in this animal model. When used as an onlay, healing is inferior to spontaneous urethral regeneration, as SIS impedes smooth muscle cell regeneration. When used as a tube, there is complete scarring and urethral luminal occlusion.

Fibroblast Growth Factor-2 Represses Platelet-derived Growth Factor Receptor-α (PDGFR-α) Transcription via ERK1/2-dependent Sp1 Phosphorylation and an Atypical cis-Acting Element in the Proximal PDGFR-α Promoter

Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant for vascular smooth muscle cells (SMCs) whose biological activity is mediated via its high affinity interaction with specific cell surface receptors. The molecular mechanisms governing the expression of PDGF receptor-alpha (PDGFR-alpha) are poorly understood. Here we demonstrate that PDGFR-alpha protein and transcriptional regulation in SMCs is under the positive regulatory influence of the zinc finger nuclear protein, Sp1. Electrophoretic mobility shift, competition, and supershift analysis revealed the existence of an atypical G-rich Sp1-binding element located in the PDGFR-alpha promoter -61 to -52 bp upstream of the transcriptional start site. Mutation of this sequence ablated endogenous Sp1 binding and activation of the PDGFR-alpha promoter. PDGFR-alpha transcription, mRNA, and protein expression were repressed in SMCs exposed to fibroblast growth factor-2 (FGF-2). This inhibition was rescued by the blockade of extracellular signal-regulated kinase-1/2 (ERK1/2). FGF-2 repression of PDGFR-alpha transcription was abrogated upon mutation of this Sp1-response element. FGF-2 stimulated Sp1 phosphorylation in an ERK1/2- but not p38-dependent manner, the growth factor enhancing Sp1 interaction with the PDGFR-alpha promoter. Mutation of residues Thr(453) and Thr(739) in Sp1 (amino acids phosphorylated by ERK) blocked FGF-2 repression of PDGFR-alpha transcription. These findings, taken together, demonstrate that FGF-2 stimulates ERK1/2-dependent Sp1 phosphorylation, thereby repressing PDGFR-alpha transcription via the -61/-52 element in the PDGFR-alpha promoter. Phosphorylation triggered by FGF-2 switches Sp1 from an activator to a repressor of PDGFR-alpha transcription, a finding previously unreported in any Sp1-dependent gene.

Gene expression profiling of resting and activated vascular smooth muscle cells by serial analysis of gene expression and clustering analysis

Migration and proliferation of vascular smooth muscle cells (SMCs) are key events in atherosclerosis. However, little is known about alterations in gene expression upon transition of the quiescent, contractile SMC to the proliferative SMC. We performed serial analysis of gene expression (SAGE) of cultured, human SMCs, either grown under resting circumstances or activated with an atherogenic stimulus. Analysis of tags, representing 47,209 and 47,259 mRNAs from a library of resting and activated SMCs, respectively, identified 105 tags induced and 52 tags repressed greater than fivefold. To evaluate the relevance in SMC biology of unmatched, regulated tags, we performed hierarchical clustering analysis, based on their expression profiles in public SAGE databases, and clustered these novel genes in distinct groups. The regulation in SMCs was confirmed by Northern blotting for representative genes of these groups. Plasminogen activator inhibitor-2 has not been associated with atherosclerosis before and was localized to atherosclerotic lesions.

TIME DEPENDENT SMOOTH MUSCLE REGENERATION AND MATURATION IN A BLADDER ACELLULAR MATRIX GRAFT: HISTOLOGICAL STUDIES AND IN VIVO FUNCTIONAL EVALUATION

We evaluated the time dependence of smooth muscle regeneration and restoration of in vivo functional properties in bladder augmented with a bladder acellular matrix graft. A total of 45 Sprague-Dawley rats underwent augmentation cystoplasty with a bladder acellular matrix graft. Two rats each were sacrificed at various intervals within the first 21 days and 6 each were sacrificed at 4, 8 and 12 weeks. This second group underwent preoperative and postoperative assessment of bladder function, including cystometry, electrostimulation and stimulation with ice water, potassium and carbachol, as well as labeling of the bladder wall by the injection of fluorescent microspheres. After sacrifice slides of the bladders prepared for hematoxylin and eosin, trichrome, KI67, vimentin, desmin, smooth muscle specific alpha-actin and fluorescent microspheres were evaluated. Within 2 weeks the number of cells in the matrix as well as the proliferation index increased rapidly and then decreased gradually. Erythrocytes and inflammatory cells were found in the matrix within 2 to 4 days, followed by fibroblasts. A bladder host-to-matrix shift was evident by the appearance of microspheres in the matrix. Cell marker expression indicated the early appearance of vimentin and alpha-actin within the first 10 days. Distinct desmin expression was observed later, when the first smooth muscle cells were recognized. Functional evaluation revealed restored bladder function at 12 weeks. The time dependent increase of muscle cell markers during smooth muscle cell regeneration in a bladder acellular matrix graft is concordant with the progressive restoration of bladder function. These results may support the bladder acellular matrix graft concept for clinical application.

ATHEROSCLEROSIS-INDUCED CHRONIC ISCHEMIA CAUSES BLADDER FIBROSIS AND NON-COMPLIANCE IN THE RABBIT

The overall goal was to determine whether chronic ischemia and hypercholesterolemia interfere with bladder function and structure. The roles of atherosclerosis-induced chronic ischemia and hypercholesterolemia in bladder fibrosis and non-compliance were studied in the rabbit. The relationship between ischemia-induced changes in the expression of transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF) and the severity of bladder fibrosis was also investigated. Male New Zealand White rabbits were divided into chronic bladder ischemia (CBI, n = 11), hypercholesterolemia (Hch, n = 8) and control (n = 8) groups. The CBI group underwent balloon endothelial injury of the iliac arteries and received a 0.5% cholesterol diet. The Hch group received a 0.5% cholesterol diet alone. The control group was placed on a regular diet. After 16 weeks, iliac artery and bladder wall blood flow measurements, cystometrograms (CMG) and aorto-iliac arteriograms were obtained in all animals. Iliac arteries and bladder tissues were processed for histological staining and computer-assisted histomorphometric image analysis. The expressions of TGF-beta1 and bFGF in bladder tissue were determined by immunohistochemical staining utilizing monoclonal antibodies. At 16 weeks, arteriography and histology showed significant diffuse atherosclerotic occlusive disease of the aorto-iliac arteries in the CBI group. Iliac artery and bladder wall blood flows were significantly decreased in the CBI group compared with the Hch and control groups. Atherosclerosis-induced CBI shifted the volume-pressure curve to the left and caused severe bladder fibrosis. Hypercholesterolemia also caused fibrosis and non-compliance but to a much lesser extent compared with those caused by CBI. In histomorphometry, the percentage of detrusor smooth muscle was moderately decreased in the Hch group and severely decreased in the CBI group compared with the control group. In immunohistochemical stains of bladder tissues, bFGF expression was similar in the three groups of animals. TGF-beta1 expression was significantly greater in bladder tissues from the CBI group compared with the Hch and control groups. Our studies show that atherosclerosis-induced chronic ischemia increases TGF-beta1 expression in the bladder leading to fibrosis, smooth muscle atrophy and non-compliance. Hypercholesterolemia also interferes with bladder structure and compliance but to a significantly lesser extent compared with CBI. Our studies suggest that arterial insufficiency and hypercholesterolemia, common aging-associated disorders, may play important roles in the pathophysiology of voiding dysfunction in the elderly.

Somatostatin sst5 inhibition of receptor mediated regeneration of rat aortic vascular smooth muscle cells.

1. The aim of the present study was to determine the effect of somatostatin (SRIF) on mitogen-induced regeneration of rat aortic vascular smooth muscle cells (VSMC) and for comparison Chinese hamster ovary (CHO)-K1 cells expressing human recombinant sst5 receptors (CHOsst5), following partial denudation of a confluent cell monolayer. Regeneration was assessed by measuring areas of recovery into the denuded area and by counting total cell numbers. 2. In VSMC, SRIF (0.1 nM - 1 microM) had no effect on the basal levels of regeneration but caused a concentration-dependent inhibition (pIC50 8.0-8.6) of the stimulated regeneration induced by submaximal concentrations of basic fibroblast growth factor (bFGF, 10 ng ml[-1]), platelet-derived growth factor-BB (PDGF, 5 ng ml[-1]) or endothelin-1 (ET-1, 100 nM). SRIF (pIC50 8.8) also inhibited bFGF-induced regeneration of CHOsst5 cells. 3. In VSMC, the inhibitory action of SRIF on the regeneration induced by bFGF (10 ng ml[-1]) was due to an anti-proliferative effect, rather than an effect on cell migration, as SRIF (0.1 nM - 1 microM) abolished bFGF-induced increases in total cell numbers. The bFGF-induced increase in cell numbers was also abolished by actinomycin D (0.1 microg ml[-1]). 4. The sst5 receptor-selective agonist, L-362,855 (pIC50 10.5), was about 100 times more potent than SRIF at inhibiting bFGF-induced regeneration of both VSMC and CHOsst5 cells whilst the sst2 receptor-selective agonist, BIM-23027 (pIC50 6.8), was approximately 20 times weaker than SRIF. 5. The sst5 receptor antagonist, BIM-23056 (100 nM), antagonized SRIF-induced inhibition of bFGF-induced regeneration in both VSMC and CHOsst5 cells (estimated pKB values 8.8 and 8.3, respectively). 6. SRIF-induced inhibition of bFGF-induced regeneration of VSMC and CHOsst5 cells was abolished by pretreating cells with pertussis toxin (100 ng ml[-1]) for 20 h. 7. These findings suggest that SRIF-induced inhibition of the proliferation of rat aortic VSMC is mediated via activation of receptors which are similar to human sst5 receptors. Furthermore this inhibitory effect is transduced via pertussis toxin-sensitive Gi/Go proteins.

Reduced smooth muscle cell regeneration in Yoshida (YOS) spontaneously hypercholesterolemic rats

Hypercholesterolemia is a predisposing factor for atherosclerosis. We studied the response to damage of vascular smooth muscle cells (SMC) from normocholesterolemic Brown Norway (BN) and from spontaneously hypercholesterolemic Yoshida (YOS) rats (16–24 month old). The regrowth rate of SMC from BN and YOS rats after freezeinduced damage was similar in the presence of fetal calf serum and of serum derived from normocholesterolemic rats, while it was reduced in the presence of serum from hypercholesterolemic rats. Freeze-injury of the abdominal aorta was followed by reduced neointima formation in YOS rats, as compared to BN rats, confirming the impaired response of vascular cells from hypercholesterolemic rats to injury. This defect may be due either to lipids or to unknown factors present in the hyperlypidemic serum.

Abundant expression of parathyroid hormone-related protein in primary rat aortic smooth muscle cells accompanies serum-induced proliferation.

Parathyroid hormone-related protein (PTHrP), which is responsible for producing hypercalcemia in patients with humoral hypercalcemia of malignancy, has recently been identified in several normal tissues. Because PTHrP, like parathyroid hormone (PTH), is known to exhibit vasodilatory properties, we investigated the expression and regulation of PTHrP mRNA in cultured rat aortic smooth muscle cells (SMC). We report here that PTHrP mRNA is expressed in SMC and is markedly induced by serum in a time- and concentration-dependent fashion. Addition of 10% fetal calf serum to serum-deprived, confluent cells, resulted in a marked induction of PTHrP mRNA by 2 h with a peak at 4-6 h. PTHrP was detected in SMC by immunocytochemistry and radioimmunoassay of conditioned medium, and was shown to be up-regulated within 24 h after the addition of serum. The serum induction of PTHrP mRNA was blocked by actinomycin D and by cycloheximide indicating the need for protein synthesis to evoke the serum effect on PTHrP gene transcription. In addition, treatment with dexamethasone, which has been previously shown to reduce the constitutive expression of PTHrP in human cancer cells, also blunted the serum induction of PTHrP mRNA in SMC. Treatment of quiescent cells with the serum mitogens platelet-derived growth factor or insulin-like growth factor-I had no effect on PTHrP, whereas the vasoactive peptides endothelin, norepinephrine and thrombin stimulated PTHrP expression. Exogenous addition of recombinant PTHrP-(1-141) had no significant effect on SMC DNA synthesis as measured by [3H]thymidine incorporation. In summary, the abundance of PTHrP mRNA and the characteristics of its regulation in SMC suggest a major role for PTHrP as a local modulator in vascular smooth muscle.

Histopathology of microvenous repair.

The histopathology of microvascular repair was investigated in the femoral vein of the rat. Previous microvascular repair studies have primarily involved arteries, giving little insight to similar processes in veins. In our study, 36 rats with an average weight of 440 gm were randomly assigned to experimental groups of 1 hour, 24 hours, 2 weeks, and 4 weeks after repair. Histological evaluation of the suture site included the use of light, scanning electron, and transmission electron microscopy. Endothelial loss was observed in all 1-hour specimens. Extensive inflammatory response appeared in 2-hour specimens. Re-endothelialization of the intima was achieved in all animals by the 2-week time interval. At 4 weeks, the animals exhibited evidence of medial regeneration of smooth muscle cells. This was predominantly occurring between adjacent suture sites. Minimal subintimal hyperplasia was localized at the suture site itself in both 2- and 4-week specimens.

An ultrastructural study of regeneration of minced smooth muscle in the vas deferens of the guinea-pig.

Electron microscopic studies were made of the regeneration of minced smooth muscle of the vas deferens of the guinea-pig 3 days to 15 weeks after operation. At 3--5 days the mince contained degenerating smooth muscle cells and dedifferentiating cells showing characteristics of embryonic smooth muscle cells: numerous free ribosomes, well developed rough endoplasmic reticulum and Golgi apparatus with few peripherally placed myofilaments associated with dense bodies. During the first two weeks of regeneration, scattered cells surrounded by debris and collagen were separated by a large extra-cellular space. After three weeks, extracellular space was reduced to near normal values. Regenerating cells had a shorter length than normal cells, but during later stages of regeneration they showed an increase in diameter. Muscle effector bundles began to form after 2 to 3 weeks. Initially there were large gaps between the muscle cells, but at later stages of bundle formation, the extracellular space between the muscle cells was much reduced. From 3 weeks, arterioles appeared between the smooth muscle bundles in the regenerating areas. Regeneration of individual smooth muscle cells was complete by 15 weeks after the operation.

Smooth muscle cell regeneration in repair of gastric anastomosis in the dog.

Tissue repair process after experimental transection of the stomach in dogs has been studied histologically and electron microscopically. Initially the incision gap is observed to be filled with loose connective tissue, in which smooth muscle cells (sm.m.c.) soon appear. On the 6th day after the operation single sm.m.c. are found dispersed among the polymorphous cell elements of the granulation tissue, comprising the area of the anastomosis. With the advance of the restorative process, increased number of sm.m.c. invides the connective tissue, replacing the defect. On the 9th and 12th days, islets of sm.m.c., often cone-shaped, adjacent with their bases to the normal muscular layer are observed in the connective tissue and within 30 to 90 days, bundles of sm.m.c., alternating with connective tissue, overbridge the operative defect and connect both edges of the transected gastric wall.