Glucose degradation products in peritoneal dialysis solution impair angiogenesis by dysregulating angiogenetic factors in endothelial and vascular smooth muscle cells.

BACKGROUND: The accumulation of glucose degradation products (GDPs) during peritoneal dialysis (PD) can lead to immature angiogenesis in the peritoneum. However, the effect of GDPs on angiogenesis, at concentrations observed in dialysate effluent, has not been widely investigated. We do not know how the inflammation observed in PD-related peritonitis affects angiogenesis of the peritoneum. METHODS: Human umbilical vessel endothelial cells (HUVEC) and human umbilical aortic smooth muscle cells (HUASMC) were used to examine the response to the three main GDPs found in peritoneal dialysate (methylglyoxal (MGO), 3-deoxyglucosone (3-DG), and 5-hydroxymethylfurfural (5-HMF). Supernatant from lipopolysaccharide (LPS)-activated murine macrophage cell lines (RAW 264.7 cells) were used to stimulate angiogenesis in the peritoneum. Changes in the expression of vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor B (PDGFB) in HUVEC, and PDGF-receptor beta (PDGF-Rß) in HUASMC, were examined by real-time PCR, Western blot, and ELISA. RESULTS: In HUVECs, the expression of PDGFB mRNA and protein were decreased by exposure to MGO, 3-DG, and 5-HMF at concentrations observed in dialysate effluent. A subsequent decrease in secreted PDGF-BB was observed. In HUASMCs, MGO and 5-HMF increased the expression of VEGF-A mRNA and protein, while 5-HMF decreased the expression of PDGF-Rß. VEGF-A is upregulated, and PDGF-Rß is downregulated, by conditioned medium of LPS-stimulated macrophages in HUASMCs. CONCLUSIONS: The GDPs of PD effluent cause an imbalance of angiogenic factors in endothelial cells and vascular smooth muscle cells that may lead to immature angiogenesis in the peritoneum.

A Real-Time, Plate-Based BRET Assay for Detection of cGMP in Primary Cells.

Cyclic guanosine monophosphate (cGMP) is a second messenger involved in the regulation of numerous physiological processes. The modulation of cGMP is important in many diseases, but reliably assaying cGMP in live cells in a plate-based format with temporal resolution is challenging. The Förster/fluorescence resonance energy transfer (FRET)-based biosensor cGES-DE5 has a high temporal resolution and high selectivity for cGMP over cAMP, so we converted it to use bioluminescence resonance energy transfer (BRET), which is more compatible with plate-based assays. This BRET variant, called CYGYEL (cyclic GMP sensor using YFP-PDE5-Rluc8), was cloned into a lentiviral vector for use across different mammalian cell types. CYGYEL was characterised in HEK293T cells using the nitric oxide donor diethylamine NONOate (DEA), where it was shown to be dynamic, reversible, and able to detect cGMP with or without the use of phosphodiesterase inhibitors. In human primary vascular endothelial and smooth muscle cells, CYGYEL successfully detected cGMP mediated through either soluble or particulate guanylate cyclase using DEA or C-type natriuretic peptide, respectively. Notably, CYGYEL detected differences in kinetics and strength of signal both between ligands and between cell types. CYGYEL remained selective for cGMP over cAMP, but this selectivity was reduced compared to cGES-DE5. CYGYEL streamlines the process of cGMP detection in plate-based assays and can be used to detect cGMP activity across a range of cell types.

CRISPR-Cas9-Mediated Epitope Tagging Provides Accurate and Versatile Assessment of Myocardin-Brief Report.

Objective- Unreliable antibodies often hinder the accurate detection of an endogenous protein, and this is particularly true for the cardiac and smooth muscle cofactor, MYOCD (myocardin). Accordingly, the mouse Myocd locus was targeted with 2 independent epitope tags for the unambiguous expression, localization, and activity of MYOCD protein. Approach and Results- 3cCRISPR (3-component clustered regularly interspaced short palindromic repeat) was used to engineer a carboxyl-terminal 3×FLAG or 3×HA epitope tag in mouse embryos. Western blotting with antibodies to each tag revealed a MYOCD protein product of ≈150 kDa, a size considerably larger than that reported in virtually all publications. MYOCD protein was most abundant in some adult smooth muscle-containing tissues with surprisingly low-level expression in the heart. Both alleles of Myocd are active in aorta because a 2-fold increase in protein was seen in mice homozygous versus heterozygous for FLAG-tagged Myocd. ChIP (chromatin immunoprecipitation)-quantitative polymerase chain reaction studies provide proof-of-principle data demonstrating the utility of this mouse line in conducting genome-wide ChIP-seq studies to ascertain the full complement of MYOCD-dependent target genes in vivo. Although FLAG-tagged MYOCD protein was undetectable in sections of adult mouse tissues, low-passaged vascular smooth muscle cells exhibited expected nuclear localization. Conclusions- This report validates new mouse models for analyzing MYOCD protein expression, localization, and binding activity in vivo and highlights the need for rigorous authentication of antibodies in biomedical research.

Inherited pathogenic mitochondrial DNA mutations and gastrointestinal stem cell populations.

Inherited mitochondrial DNA (mtDNA) mutations cause mitochondrial disease, but mtDNA mutations also occur somatically and accumulate during ageing. Studies have shown that the mutation load of some inherited mtDNA mutations decreases over time in blood, suggesting selection against the mutation. However, it is unknown whether such selection occurs in other mitotic tissues, and where it occurs within the tissue. Gastrointestinal epithelium is a canonical mitotic tissue rapidly renewed by stem cells. Intestinal crypts (epithelium) undergo monoclonal conversion with a single stem cell taking over the niche and producing progeny. We show: (1) that there is a significantly lower mtDNA mutation load in the mitotic epithelium of the gastrointestinal tract when compared to the smooth muscle in the same tissue in patients with the pathogenic m.3243A>G and m.8344A>G mutations; (2) that there is considerable variation seen in individual crypts, suggesting changes in the stem cell population; (3) that this lower mutation load is reflected in the absence of a defect in oxidative phosphorylation in the epithelium. This suggests that there is selection against inherited mtDNA mutations in the gastrointestinal stem cells that is in marked contrast to the somatic mtDNA mutations that accumulate with age in epithelial stem cells leading to a biochemical defect. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Histopathological and proteomic analyses identify integrin-ß1 as a potential mediator of phlebosclerosis in uremic patients.

BACKGROUND: Patients with uremia have an excessive mortality from cardiovascular disease (CVD). Arterial remodeling is mainly responsible for uremia-induced CVD and has been well studied, yet venous remodeling is poorly understood. Here we investigate the histopathology and proteomic profiles of venous remodeling in uremic patients. METHODS: Forearm cephalic veins were isolated from nine uremic patients during surgeries for arteriovenous fistula, and from nine healthy controls when applying surgical debridement. Hematoxylin-eosin, Masson's trichrome, von Kossa, and immunohistochemistry (IHC) against proliferating cell nuclear antigen were stained for histopathology. Isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis was executed to explore the proteome of the veins. The core regulatory protein was validated by western blot, IHC, and immunofluorescence. RESULTS: Phlebosclerosis, characterized by intimal rarefaction and medial thickening with disordered proliferation of vascular smooth muscle cells (VSMCs), was the prominent pathological manifestation of peripheral veins in uremic patients, while inflammatory cell infiltration, atherosclerosis or calcification were not obviously detected. iTRAQ analysis showed that 350 proteins were significantly changed in phlebosclerosis of uremic patients compared with healthy controls, of which integrin-ß1 (ITGß1) exhibited the strongest regulatory ability by intermolecular interaction network analysis. The enhanced ITGß1 expression was mainly co-expressed with the disordered proliferation of VSMCs while a little with vascular endothelial cells in the forearm cephalic veins of uremic patients. CONCLUSIONS: Phlebosclerosis is the prominent pathological manifestation in peripheral veins of uremic patients. This pathological alteration mainly attributes to the disordered proliferation of VSMCs, which is potentially mediated by ITGß1.

KV channel trafficking and control of vascular tone.

Membrane potential is a principal regulator of arterial contractility. Arterial smooth muscle cells express several different types of ion channel that control membrane potential, including KV channels. KV channel activation leads to membrane hyperpolarization, resulting in inhibition of voltage-dependent Ca2+ channels, a reduction in [Ca2+ ]i , and vasodilation. In contrast, KV channel inhibition leads to membrane depolarization and vasoconstriction. The ability of KV channels to regulate arterial contractility is dependent upon the number of plasma membrane-resident channels and their open probability. Here, we will discuss mechanisms that alter the surface abundance of KV channel proteins in arterial smooth muscle cells and the functional consequences of such regulation. Cellular processes that will be described include those that modulate KV channel transcription, retrograde and anterograde trafficking, and protein degradation.

Distribution of extracellular matrix molecules in human uterine tubes during the menstrual cycle: a histological and immunohistochemical analysis.

The uterine tube (UT) is an important and complex organ of the women's reproductive system. In general, the anatomy and basic histology of this organ are well-known. However, the composition and function of the extracellular matrix (ECM) of the UT is still poorly understood. The ECM is a complex supramolecular material produced by cells which is commonly restricted to the basement membrane and interstitial spaces. ECM molecules play not only a structural role, they are also important for cell growth, survival and differentiation in all tissues. In this context, the aim of this study was to evaluate the deposition and distribution of type I and III collagens and proteoglycans (decorin, biglycan, fibromodulin and versican) in human UT during the follicular and luteal phases by using histochemical and immunohistochemical techniques. Our results showed a broad synthesis of collagens (I and III) in the stroma of the UT. The analysis by regions showed, in the mucosa, a specific distribution of versican and fibromodulin in the epithelial surface, whereas decorin and fibromodulin were observed in the lamina propria. Versican and decorin were found in the stroma of the muscular layer, whereas all studied proteoglycans were identified in the serosa. Curiously, biglycan was restricted to the wall of the blood vessels of the serosa and muscular layers. Furthermore, there was an immunoreaction for collagens, decorin, versican and fibromodulin in the UT peripheral nerves. The differential distribution of these ECM molecules in the different layers of the UT could be related to specific structural and/or biomechanical functions needed for the oviductal transport, successful fertilization and early embryogenesis. However, further molecular studies under physiological and pathological conditions are still needed to elucidate the specific role of each molecule in the human UT.

Altered Integrins Expression of Patients Affected by Cryptorchidism.

OBJECTIVES: The study aimed to investigate the expression of the integrin isoforms α7A and ß1A, expressed by myogenic precursor cells, and α7B and ß1D, expressed by mature muscle cells in the cremaster of patients affected by an undescended testis. METHODS: Fifteen samples of cremaster were obtained from patients undergoing surgery for an undescended testis. Thirty control specimens of cremaster were harvested from patients with congenital hydrocele or inguinal hernia. Immunofluorescent analysis was carried out using anti-α7A, ß1A, α7B, and ß1D integrin antibodies. Sections were observed using confocal laser scanning microscopy. RESULTS: As compared with controls, a significant loss of a α7B (p = 0.0355) and ß1D (p = 0.0069) integrins and a higher expression of α7A (p = 0.0003) and ß1A (p = 0.0150) was detected in the cremaster of patients affected by an undescended testis. CONCLUSIONS: Our data document a critical alteration of the cytoskeleton of cremasteric smooth muscle cells in patients with an undescended testis. This might explain the altered function in smooth muscle cells in cremaster implied during testicular descent. We therefore speculate that the postnatal splicing of α7A to α7B and of ß1A to ß1D integrins is delayed. This could account for the common clinical scenario of spontaneous descent of the testes in the first months of life.

Heterogeneous histomorphology, yet homogeneous vascular smooth muscle cell dedifferentiation, characterize human aneurysm disease.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a frequent, potentially life-threatening, disease that can only be treated by surgical means such as open surgery or endovascular repair. No alternative treatment is currently available, and despite expanding knowledge about the pathomechanism, clinical trials on medical aneurysm abrogation have led to inconclusive results. The heterogeneity of human AAA based on histologic examination is thereby generally neglected. In this study we aimed to further elucidate the role of these differences in aneurysm disease. METHODS: Tissue samples from AAA and popliteal artery aneurysm patients were examined by histomorphologic analysis, immunohistochemistry, Western blot, and polymerase chain reaction. The results were correlated with clinical data such as aneurysm diameter and laboratory results. RESULTS: The morphology of human AAA vessel wall probes varies tremendously based on the grade of inflammation. This correlates with increasing intima/media thickness and upregulation of the vascular endothelial growth factor cascade but not with any clinical parameter or the aneurysm diameter. The phenotypic switch of vascular smooth muscle cells occurred regardless of the inflammatory state and expressional changes of the transcription factors Kruppel-like factor-4 and transforming growth factor-ß lead to differential protein localization in aneurysmal compared with control arteries. These changes were in similar manner also observed in samples from popliteal artery aneurysms, which, however, showed a more homogenous phenotype. CONCLUSIONS: Heterogeneity of AAA vessel walls based on inflammatory morphology does not correlate with AAA diameter yet harbors specific implications for basic research and possible aneurysm detection.

Cutaneous/subcutaneous mesenchymal proliferations of the breast.

Cutaneous mesenchymal "spindle cell" lesions arising in the vicinity of the breast represent a complex clinical and diagnostic scenario which may overlap histopathologically and immunohistochemically with mammary spindle cell proliferations, potentially impacting management and overall prognostication. In this review, we suggest a pattern-based approach to assist in the evaluation of these lesions. A comprehensive description of each entity is accompanied by a cutaneous and mammary differential diagnosis.

[Investigation of the structure of a functionally intact xenopericardial bioconduit after long-term implantation]. / Issledovanie struktury funktsional'no sokhrannogo ksenoperikardial'nogo bioproteza posle prodolzhitel'nogo perioda implantatsii.

AIM: to investigate the cellular composition of a functionally intact xenopericardial valve in a recipient with acquired mitral defect after long-term implantation. MATERIAL AND METHODS: A Uniline bioconduit (BC) ('Neocor', Kemerovo) removed from the heart in the mitral position at 7.2 years after implantation was investigated. Heart valve leaflets were fixed in a buffered 4% paraformaldehyde solution and imbedded in paraffin or epoxy resin. Slices made from the paraffin samples were stained with hematoxylin and eosin or underwent immunohistochemical (IHC) examination for typing endothelial cells, smooth muscle cells, macrophages, fibroblasts, and T and B lymphocytes. The epoxy resin-embedded samples were examined using light and scanning electron microscopy according to the original procedure. For this, the samples were ground and polished, then stained with toluidine blue and basic fuchsin or contrasted with uranyl acetate and lead citrate. RESULTS: Different cell types were found in the outer layers of heart valve leaflets. IHC showed that endothelial cells, macrophages, smooth muscle cells, and fibroblasts were present in the samples. A relationship was found between the degree of degenerative changes in the BC surface and the magnitude of cellular infiltration in xenotissue. This paper debates whether impaired integrity of the surface leaflet layers plays a trigger role in structural dysfunctions of the implanted valves and whether BC endothelialization has a protective effect, which can considerably reduce the immunogenicity of xenotussie and prevent the penetration of recipient cells. CONCLUSION: The paper shows that it is expedient to modify the surface of the heart valve leaflets in order to create favorable conditions for the attachment and function of endothelial progenitor cells.

Deficiency of endothelium-specific transcription factor Sox17 induces intracranial aneurysm.

BACKGROUND: Intracranial aneurysm (IA) is a common vascular disorder that frequently leads to fatal vascular rupture. Although various acquired risk factors associated with IA have been identified, the hereditary basis of IA remains poorly understood. As a result, genetically modified animals accurately modeling IA and related pathogenesis have been lacking, and subsequent drug development has been delayed. METHODS AND RESULTS: The transcription factor Sox17 is robustly expressed in endothelial cells of normal intracerebral arteries. The combination of Sox17 deficiency and angiotensin II infusion in mice induces vascular abnormalities closely resembling the cardinal features of IA such as luminal dilation, wall thinning, tortuosity, and subarachnoid hemorrhages. This combination impairs junctional assembly, cell-matrix adhesion, regeneration capacity, and paracrine secretion in endothelial cells of intracerebral arteries, highlighting key endothelial dysfunctions that lead to IA pathogenesis. Moreover, human IA samples showed reduced Sox17 expression and impaired endothelial integrity, further strengthening the applicability of this animal model to clinical settings. CONCLUSIONS: Our findings demonstrate that Sox17 deficiency in mouse can induce IA under hypertensive conditions, suggesting Sox17 deficiency as a potential genetic factor for IA formation. The Sox17-deficient mouse model provides a novel platform to develop therapeutics for incurable IA.

[Dynamic alteration of microRNA in high phosphorus induced calcification of vascular smooth muscle cell].

OBJECTIVE: To study the change of microRNA during the early stage of high phosphorus induced vascular smooth muscle cell (VSMC) calcification and its related mechanism. METHODS: The in vitro calcification model was created through stimulating VSMC cell line A7r5 with high Pi (2.6 mmol/L) for 7 d. The calcification was validated through ocresolphthalein complexone colorimetry to detect the cellular calcium content, real-time PCR to measure the calcification-related gene expression and alizarin red staining to observe the formation of calcium nodules. Based on the cell calcification model, microRNA microarray array was applied to screen the profiles of microRNA expression in VSMC following high Pi stimulation for different periods (0, 3 and 12 h). The array data were analyzed by TAM tool to explore the activated signaling pathway. RESULTS: The calcium content of A7r5 cells induced by high Pi was increased 9.6 times high as cells without Pi treatment (P<0.05). VSMC contractile phenotype genes (SM-α actin, SM22) were down-regulated (P<0.05), while calcification-related genes (BMP2, MSX2, Runx2) were up-regulated (P<0.05) in VSMC stimulated by high Pi. The calcium nodules were obviously formed in cells after 7 d high Pi treatment. In microarray experiment, 680 individual microRNAs were detected in high Pi-treated VSMCs at different time points (0, 3 and 12 h). Among these genes, miR-183, miR-664 and miR-9* were increased whereas miR-542-5P, let-7f and miR-29a were decreased in time-dependent manners. Twenty-six kinds of signaling pathways, including cell apoptosis, differentiation and proliferation, were significantly activated. All these activated pathways were associated with calcification. CONCLUSION: This study implies that microRNA changed in high Pi-induced VSMCs may involve in the process of calcification.

[Biomarkers for Atherosclerotic Diseases: Significance of Intimal Smooth Muscle Cell-Derived Circulating Marker].

Vascular cells with the active molecules they release are involved in the progression of atherosclerosis. These cell-related circulating markers are expected to reflect the pathological conditions of atherosclerotic lesions. In response to stimuli from other cells or active molecules, smooth muscle cells (SMCs) in the medial layer change their phenotype from contractile to synthetic, migrate from the media into the intima, and there they proliferate and release various kinds of cytokines, proteinases, and extra-cellular matrices. Thus, the functions of intimal SMCs are believed to play key roles in plaque formation. The incomplete or sustained activation of intimal SMCs may cause the fragility of plaques under pathological conditions, i.e., diabetes and dyslipidemia. LR11 is one of the genes specifically expressed in intimal SMCs, but not in me- dial SMCs, and it increases the sensitivity of intimal SMCs to migration in response to cytokines including angiotensin II. The soluble form of LR11 is in circulation, and the levels increase transiently after coronary intervention in the period corresponding to the migration of SMCs. The increase in initial sLR11 levels is negatively correlated with event-free survival for CVD endpoints. These results suggest that biomarkers reflecting the pathological conditions of intimal SMCs may be useful as surrogate and/or companion markers for the treatment of atherosclerotic diseases. [Review].

Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC-MS/MS. 1. Preparation of more than 4000 native protein maps.

Soluble proteins of human bronchial smooth muscle cells (HBSMC) were separated by nondenaturing micro 2DE and a 30 mm × 40 mm area of the CBB-stained slab gel (1.0 mm thick) was cut into 1.1 mm × 1.1 mm squares, then the proteins in the 972 gel pieces (squares) were applied to quantitative LC-MS/MS. Grid-cutting of the gel was employed to; (i) ensure the total analysis of the proteins in the area, (ii) standardize the conditions of analysis by LC-MS/MS, (iii) reconstruct the protein distribution patterns from the quantity data [1]. Totally 4323 proteins were identified in successfully analyzed 967 squares and the quantity distribution of each was reconstructed as a color density pattern (a native protein map). The quantity of the proteins distributed from 3.6% to 1 × 10(-5) % of the total protein quantity in the grid area. Each protein map was characterized by several features, including the position of quantity peak square, number of detected squares, and degree of concentration (focused or dispersed). About 4% of the proteins were detected in 100 or more squares, suggesting that they might be ubiquitous and interacting with other proteins. In contrast, many proteins showed more concentrated quantity distribution and the quantity peak positions of 565 proteins with a defined degree of concentration were summarized into a quantity peak map. These results for the first time visualized the distribution patterns of cellular proteins on a nondenaturing 2D gel.

Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC-MS/MS. 2. Similarity search between protein maps for the analysis of protein complexes.

Human bronchial smooth muscle cell soluble proteins were analyzed by a combined method of nondenaturing micro 2DE, grid gel-cutting, and quantitative LC-MS/MS and a native protein map was prepared for each of the identified 4323 proteins [1]. A method to evaluate the degree of similarity between the protein maps was developed since we expected the proteins comprising a protein complex would be separated together under nondenaturing conditions. The following procedure was employed using Excel macros; (i) maps that have three or more squares with protein quantity data were selected (2328 maps), (ii) within each map, the quantity values of the squares were normalized setting the highest value to be 1.0, (iii) in comparing a map with another map, the smaller normalized quantity in two corresponding squares was taken and summed throughout the map to give an "overlap score," (iv) each map was compared against all the 2328 maps and the largest overlap score, obtained when a map was compared with itself, was set to be 1.0 thus providing 2328 "overlap factors," (v) step (iv) was repeated for all maps providing 2328 × 2328 matrix of overlap factors. From the matrix, protein pairs that showed overlap factors above 0.65 from both protein sides were selected (431 protein pairs). Each protein pair was searched in a database (UniProtKB) on complex formation and 301 protein pairs, which comprise 35 protein complexes, were found to be documented. These results demonstrated that native protein maps and their similarity search would enable simultaneous analysis of multiple protein complexes in cells.

Proinflammatory role of stem cells in abdominal aortic aneurysms.

OBJECTIVE: The pathogenesis of abdominal aortic aneurysm (AAA) formation includes inflammation, vascular smooth muscle cell apoptosis, extracellular matrix degradation, and oxidative stress. That multipotent stem cells have an important role in cardiovascular health and disease has been well established, but the role of stem cells in aortic structural deterioration is poorly defined. We sought to describe the presence of stem cells in human AAA tissue and also investigated the differentiation of stem cells within the aneurysmal aorta. METHODS: Infrarenal aortic wall specimens were collected from patients (n = 7) undergoing open AAA surgical repair. Nonaneurysmal infrarenal aortic control samples (n = 4) were collected at autopsies. Using immunohistochemistry, we compared the abundance of Stro1-positive ((+)), c-kit(+), and CD34(+) cells in aortic tissue. Using double-immunofluorescence staining, we evaluated stem cell differentiation into smooth muscle cells (SM22), fibroblasts (FSP1), and macrophages (CD68). We then investigated the colocalization of CD68(+) cells with the cellular marker of proliferation Ki67. RESULTS: The media and adventitia of infrarenal AAA samples both demonstrated a significantly greater number of c-kit(+) and CD34(+) cells compared with matched control nonaneurysmal aortic tissues; however, the abundance of Stro1(+) cells was not significantly different between the groups. Using double-immunofluorescence staining, we identified that AAA stem cells express the macrophage marker CD68 but not the smooth muscle cell marker SM22 or the fibroblast marker FSP1. CD68(+) cells within the aortic wall colocalized with the cellular marker of proliferation Ki67. CONCLUSIONS: Stem cells are significantly elevated in infrarenal AAA tissue compared with matched control aortic tissue. Our data also demonstrate that AAA stem cells express macrophage surface antigens but not smooth muscle cell or fibroblast markers. Furthermore, CD68(+) cells within the aortic wall colocalized with the cellular marker of proliferation Ki67. These finding suggest an inflammatory/immune role of stem cells during AAA pathogenesis and raise the possibility of localized replenishment therapy within the aneurysm wall.

Intranuclear expression of progesterone receptors in smooth muscle cells of renovascular fibromuscular dysplasia: a pilot study.

BACKGROUND: The pathogenesis of fibromuscular dysplasia (FMD) remains poorly understood. Yet, understanding this mechanism has taken on new urgency after recent evidence indicating that FMD is not as rare as previously thought. We speculated that hormonal receptors in the walls of dysplastic renal arteries were implicated in the pathogenesis of FMD. METHODS: We undertook a pilot prospective case-control study comparing histologic findings from renal arteries that were surgically removed in 2 patient groups. The case group included 6 samples from FMD patients who underwent surgery for stenosis or aneurysm caused by FMD. The control group included 3 FMD-free patients who underwent nephrectomy for nonvascular causes. Surgical specimens were sent to the histology laboratory. FMD was defined preoperatively using conventional radiologic criteria and was confirmed by histologic examination. RESULTS: Immunohistochemical staining detected intense progesterone receptor expression in the nuclei of smooth muscle cells in FMD patients. No progesterone receptor expression was found in the FMD-free patients. Estrogen receptor expression was not noted in the 2 groups. CONCLUSIONS: This preliminary finding may suggest that progesterone plays a key role in the pathogenesis of FMD and opens the fields of genetic and therapeutic approaches.

Intracellular calcium concentration of corpus cavernosum smooth muscle cells is decreased by the overexpression of PnNOS gene in adipose tissue-derived stem cells.

The study investigated the effects of adipose tissue-derived stem cells (ADSCs) modified with penile neuronal nitric oxide synthase (PnNOS) gene on intracellular calcium concentration in rat corpus cavernosum smooth muscle cells (CCSMCs). ADSCs and CCSMCs of Sprague-Dawley (SD) rats were isolated and cultured in vitro respectively. The rat PnNOS gene was transferred into the ADSCs mediated by a recombinant adenovirus vector. The expression of the PnNOS gene was detected. At the same time, the concentration of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) was assayed. After coculturing with the CCSMCs of SD rats, which were isolated and expanded ex vivo, the cGMP and NO levels of ADSCs and CCSMCs were measured. Intracellular calcium concentration ([Ca(2+) ]i ) in rat CCSMCs was measured with Fluo-3/AM by flow cytometer after cocultured with ADSCs overexpressing PnNOS gene. The mRNA and protein expression of PnNOS gene mediated by recombinant adenovirus vector significantly overexpressed and lasted at least 2 weeks. Meanwhile, the concentration of NO and cGMP in ADSCs was greatly increased. The concentration of cGMP was significantly increased, and [Ca(2+) ]i was obviously decreased in CCSMCs compared with the control groups (P < 0.05) after cocultured with ADSCs for 3 days. These findings demonstrated that ADSCs overexpressing PnNOS gene might decrease [Ca(2+) ]i in CCSMCs by up-regulating NO-cGMP signalling pathway.

Pannexin1 regulates α1-adrenergic receptor- mediated vasoconstriction.

RATIONALE: The coordination of vascular smooth muscle cell constriction plays an important role in vascular function, such as regulation of blood pressure; however, the mechanism responsible for vascular smooth muscle cell communication is not clear in the resistance vasculature. Pannexins (Panx) are purine-releasing channels permeable to the vasoconstrictor ATP and thus may play a role in the coordination of vascular smooth muscle cell constriction. OBJECTIVE: We investigated the role of pannexins in phenylephrine- and KCl-mediated constriction of resistance arteries. METHODS AND RESULTS: Western blot, immunohistochemistry, and immunogold labeling coupled to scanning and transmission electron microscopy revealed the presence of Panx1 but not Panx2 or Panx3 in thoracodorsal resistance arteries. Functionally, the contractile response of pressurized thoracodorsal resistance arteries to phenylephrine was decreased significantly by multiple Panx inhibitors (mefloquine, probenecid, and (10)Panx1), ectonucleotidase (apyrase), and purinergic receptor inhibitors (suramin and reactive blue-2). Electroporation of thoracodorsal resistance arteries with either Panx1-green fluorescent protein or Panx1 small interfering RNA showed enhanced and decreased constriction, respectively, in response to phenylephrine. Lastly, the Panx inhibitors did not alter constriction in response to KCl. This result is consistent with coimmunoprecipitation experiments from thoracodorsal resistance arteries, which suggested an association between Panx1 and α1D-adrenergic receptor. CONCLUSIONS: Our data demonstrate for the first time a key role for Panx1 in resistance arteries by contributing to the coordination of vascular smooth muscle cell constriction and possibly to the regulation of blood pressure.