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Cell-mediated cardiac regeneration remains a challenge as a therapeutic option in heart failure, but modest success using experimental models suggests that a better understanding of normal histogenesis will be needed to make progress towards cardiac regeneration. Recent studies of the heart show that the interstitium informs organogenesis and responsiveness to pathological stimuli through continuous bidirectional cross-talk between cardiomyocytes and non-cardiac cells. Here, we introduce the concept of the “cardiovascular unit” (CVU) as a building block of the heart, which includes cardiomyocytes and adjacent capillaries and fibroblasts. We discuss how the CVU might be used as a tool for re-interpreting degenerative changes of the myocardium during aging and hypertrophy, and might represent the hallmark for successful cell therapy strategies in cardiac regeneration. [Copyright &y& Elsevier]

Different vertebrate species have different cardiac regeneration rates: high in teleost fish, moderate in urodele amphibians, and almost negligible in mammals. Regeneration may occur through stem and progenitor cell differentiation or via dedifferentiation with residual cardiomyocytes reentering the cell cycle. In this review, we will examine the ability of zebrafish and newts to respond to cardiac damage with de novo cardiogenesis, whereas rodents and humans respond with a marked fibrogenic response and virtually no cardiomyocyte regeneration. Concerted strategies are needed to overcome this evolutionarily imposed barrier and optimize cardiac regeneration in mammals. [ABSTRACT FROM AUTHOR]

The article focuses on the mechanisms of endothelial progenitor cell (EPC) reduction and dysfunction associated with diabetes as well as their role in diabetes complications. It states that type 1 and type 2 diabetic patients have less circulating EPC than matched healthy subjects based on both cytometric and culture methods. It mentions that the binomial insulin resistance/hyperinsulinemia is another possible link between diabetes and EPC alterations.

Abstract: Objective: Transplantation of stem cells in the acute ischemic myocardium (AMI) may play a role in the recovery of cardiac function. Here, we investigated the ability of amniotic fluid-derived mesenchymal cells (AFC) for phenotypic conversion to vascular cells and cardiomyocytes (CM) when autotransplanted in a porcine model of AMI. Methods: Single AFC preparations were taken from 12 fetuses 3 days before normal delivery. AFC were expanded in vitro and stored separately until animals of the original litter weighed 22–25kg. A new model of AMI, i.e. 45-min circumflex coronary occlusion followed by wall dissection, was used to assess AFC differentiation potential. CMFDA-labeled AFC were autogenically transplanted in the ischemic area 1 week after AMI induction. Thirty days later, pigs were sacrificed and the phenotypic profile of transplanted AFC was assessed and compared to the corresponding pre-injection pattern. Results: AFC showed in vitro to be of mesenchymal type also expressing markers of ‘embryonic stem’ cells (SSEA4 and Oct-4), as well as endothelial (von Willebrand factor, VE-cadherin) and smooth muscle (SM α-actin, SM22) cells. Thirty days after transplantation, in the survived AFC (5±1%) ‘embryonic stem’ cell markers disappeared and mesenchymal cell markers were down regulated with the exception of smooth muscle and endothelial antigens. No evidence for expression of cardiac troponin I was found.Conclusions: In the conditions used in this study, AFC were able to transdifferentiate to cells of vascular cell lineages but not to CM. Thus, porcine AFC may require further ex vivo re-programming to be suitable for therapeutic use in AMI. [Copyright &y& Elsevier]

The myosin heavy-chain composition of human smooth muscle has been investigated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, enzyme immunoassay, and enzyme-immunoblotfing procedures. A polyclonal and a monoclonal antibody specific for smooth muscle myosin heavy chains were used in this study. The two antibodies were unreactive with sarcomeric myosin heavy chains and with platelet myosin heavy chain on enzyme immunoassay and immunoblots, and stained smooth muscle cells but not non-muscle cells in cryosections and cultures processed for indirect immunofluorescence. Two myosin heavy-chain isoforms, designated MHC-1 and MHC-2 (205 kDa and 200 kDa, respectively) were reactive with both antibodies on immunoblots of pyrophosphate extracts from different smooth muscles (arteries, veins, intestinal wall, myometrium) electrophoresed in 4% polyacrylamide gels. In the pulmonary artery, a third myosin heavy-chain isoform (MHC-3, 190 kDa) electrophoretically and antigenically distinguishable from human platelet myosin heavy chain, was specifically recognized by the monoclonal antibody. Analysis of muscle samples, directly solubilized in a sodium dodecyl sulfate solution, and degradation experiments performed on pyrophosphate extracts ruled out the possibility that MHC-3 is a proteolyfic artefact. Polypeptides of identical electrophoretic mobility were also present in the other smooth muscle preparations, but were unreactive with this antibody.
The presence of three myosin heavy-chain isoforms in the pulmonary artery may be related to the unique physiological properties displayed by the smooth muscle of this artery. [ABSTRACT FROM AUTHOR]

Presents a study which investigated the effects of retinal and peripheral vascular complications on circulating progenitor cells in type 2 diabetic patients. Research design and methods; Analysis of peripheral blood progenitor cells for surface antigen expression; Technique used in confirming the enhanced endothelial differentiation of circulating progenitors.

Abstract: Atherosclerotic diseases are responsible for a significant part of morbidity and mortality in western countries. According to the classical views, atherosclerotic lesions develop as the result of an inflammatory process initiated by endothelial damage. The discovery that bone marrow-derived cells participate in endothelial repair and new vessel growth has changed the pathogenetic models of cardiovascular disease. These cells, termed endothelial progenitor cells (EPCs), represent the endogenous endothelial regenerative capacity and the ability to form new collateral vessels. In this review we describe how quantitative and qualitative alterations of EPCs have a significant role in virtually all stages of the atherosclerotic process and in the clinical manifestations of the diseases: starting from the impact of risk factors on EPCs, through the mechanisms that link EPC reduction/dysfunction to plaque formation, and finally to the clinical syndromes. An attempt to diverge our attention from the vessel wall to the bloodstream reveals a central role of EPCs in atherogenesis. [Copyright &y& Elsevier]

Cell therapy has developed as a complementary treatment for myocardial regeneration. While both autologous and allogeneic uses have been advocated, the ideal candidate has not been identified yet. Amniotic fluid-derived stem (AFS) cells are potentially a promising resource for cell therapy and tissue engineering of myocardial injuries. However, no information is available regarding their use in an allogeneic context. c-kit-sorted, GFP-positive rat AFS (GFP-rAFS) cells and neonatal rat cardiomyocytes (rCMs) were characterized by cytocentrifugation and flow cytometry for the expression of mesenchymal, embryonic and cell lineage-specific antigens. The activation of the myocardial gene program in GFP-rAFS cells was induced by co-culture with rCMs. The stem cell differentiation was evaluated using immunofluorescence, RT-PCR and single cell electrophysiology. The in vivo potential of Endorem-labeled GFP-rAFS cells for myocardial repair was studied by transplantation in the heart of animals with ischemia/reperfusion injury (I/R), monitored by magnetic resonance imaging (MRI). Three weeks after injection a small number of GFP-rAFS cells acquired an endothelial or smooth muscle phenotype and to a lesser extent CMs. Despite the low GFP-rAFS cells count in the heart, there was still an improvement of ejection fraction as measured by MRI. rAFS cells have the in vitro propensity to acquire a cardiomyogenic phenotype and to preserve cardiac function, even if their potential may be limited by poor survival in an allogeneic setting. [ABSTRACT FROM AUTHOR]

Abstract: The potential for in vitro colonization of decellularized valves by human bone marrow mesenchymal stem cells (hBM-MSCs) towards the anisotropic layers ventricularis and fibrosa and in homo- vs. heterotypic cell–ECM interactions has never been investigated. hBM-MSCs were expanded and characterized by immunofluorescence and FACS analysis. Porcine and human pulmonary valve leaflets (p- and hPVLs, respectively) underwent decellularization with Triton X100–sodium cholate treatment (TRICOL), followed by nuclear fragment removal. hBM-MSCs (2×106 cells/cm2) were seeded onto fibrosa (FS) or ventricularis (VS) of decellularized PVLs, precoated with FBS and fibronectin, and statically cultured for 30 days. Bioengineered PVLs revealed no histopathological features but a reconstructed endothelium lining and the presence of fibroblasts, myofibroblasts and SMCs, as in the corresponding native leaflet. The two valve layers behaved differently as regards hBM-MSC repopulation potential, however, with a higher degree of 3D spreading and differentiation in VS than in FS samples, and with enhanced cell survival and colonization effects in the homotypic ventricularis matrix, suggesting that hBM-MSC phenotypic conversion is strongly influenced in vitro by the anisotropic valve microstructure and species-specific matching between extracellular matrix and donor cells. These findings are of particular relevance to in vivo future applications of valve tissue engineering. [Copyright &y& Elsevier]

Abstract: In the last 10 years an increasing interest has been devoted to the study of endothelial progenitor cells (EPCs), a subtype of immature cells involved in endothelial repair and neoangiogenesis. EPCs have been discovered as a novel integrated part of the cardiovascular system, which plays a comprehensive role in tissue homeostasis. Consistently, alterations and/or reduction of the circulating EPC pool have been associated with different manifestations of cardiovascular disorders and atherosclerosis. This is why, the extent of the EPC pool is now considered a mirror of vascular health, while EPC reduction has become a surrogate biomarker of cardiovascular risk and of the ongoing vascular damage. Unfortunately, the methods used to study EPCs still lack standardization, and this is significantly decelerating progress in the field. In this review, we focus on some aspects related to the two methods used to assess circulating EPCs: flow cytometry and cell culture. We uncover the many traps hidden in the choice of the right protocol, and suggest the best solutions on the basis of evidence and background theories. [Copyright &y& Elsevier]

Abstract: The potential of collagen scaffolds for promoting angiogenesis/arteriogenesis was studied in vivo by implantation on healthy or cryoinjured left ventricles of rats up to 60 days post-injury. Blood vessels content and extra-vascular cell infiltration were evaluated within the collagen scaffold, the cryoinjured areas, and the “border zones” of the myocardium facing the cryoinjured zones. The collagen cardiac patches were almost completely absorbed in 60 days and became populated by new arterioles and capillaries in both intact and cryoinjured heart (arterioles in cryoinjured vs. intact zones were about 2,3-fold higher; capillaries in cryoinjured vs. intact zones were 1.7-fold higher). Collagen cardiac patches exerted a “trophic” effect on the organizing granulation tissue that emerged from the wound-healing process, increasing vessel density of 2.7-fold for arterioles and 4-fold for capillaries. Interstitial cells in collagen cardiac patches rarely (<1%) expressed cardiogenic stem cells markers such as Sca-1- or MDR1, whereas markers of neural crest cells GFAP+/nestin+ cells ranged from 3/30% to 30/70% in collagen cardiac patches placed on intact vs. cryoinjured heart, respectively. Myofibroblasts and cardiomyocytes (CM) were absent but macrophages populated the collagen scaffolds even after 60 days from implantation. Western blotting of collagen cardiac patches after implantation on intact/cryoinjured hearts confirmed that markers of endothelial and smooth muscle cells, but not of CM, were expressed. The porous collagen scaffold was able to elicit a powerful angiogenetic and arteriogenetic response in the intact and cryoinjured hearts, representing an ideal tool for therapeutic angio-arteriogenesis and a potentially useful substrate for stem cell seeding. [Copyright &y& Elsevier]

Abstract: Human amniotic fluid-derived stem (AFS) cells, similarly to embryonic stem cells, could possess privileged immunological characteristics suitable for a successful transplantation even in a discordant xenograft system. We investigated whether AFS cells could be fruitfully used in a rat model of myocardial infarction. c-kit immunomagnetic-sorted AFS cells were characterized by flow cytometric analysis and cytospins as well as reverse-transcription polymerase chain reaction, Western blotting and immunocytochemistry for cardiovascular differentiation markers. In vitro, AFS cell phenotypic conversion was assayed by cardiovascular-specific induction media or co-cultured with rat neonatal cardiomyocytes. AFS cells showed mRNAs and/or protein for endothelial (angiopoietin, CD146) and smooth muscle (smoothelin) cells, and cardiomyocyte (Nkx2.5, MLC-2v, GATA-4, β-MyHC) markers. Acquisition of a cardiomyocyte-like phenotype in rare AFS cells could be seen only in co-cultures with rat neonatal cells. In vivo, AFS cells xenotransplantated in a rat model of myocardial infarction, with or without cyclosporine treatment, or in intact heart from immuno-competent or immuno-deficient animals were acutely rejected due to the different recruitment of recipient CD4+, CD8+ T and B lymphocytes, NK cells and macrophages. This reaction is most likely to be linked to expression of B7 co-stimulatory molecules CD80 and CD86 as well as macrophage marker CD68 on AFS cells. Xenotransplanted AFS cells gave also rise in some animals to cell masses in the subendocardium and myocardium suggestive of a process of chondro-osteogenic differentiation. Despite AFS cells in vitro can differentiate to some extent to cells of cardiovascular lineages, their in vivo use in xenotransplantation for cell therapy of myocardial infarction is hampered by their peculiar immunogenic properties and phenotypic instability. [Copyright &y& Elsevier]

The existence of specific differentiation markers for arterial smooth muscle (SM) cells is still a matter of debate. A clone named MM1 was isolated from a library of monoclonal antibodies to adult porcine aorta, which in vivo binds to arterial but not venous SM cells, except for the pulmonary vein. MM1 immunoreactivity in Western blotting involved bands in the range of Mr 33–226 kDa, in both arterial and venous SM tissues. However, immunoprecipitation experiments revealed that MM1 bound to a 100-kDa polypeptide that was present only in the arterial SM extract. By mass spectrometry analysis of tryptic digests from MM1-positive 130- and 120-kDa polypeptides of aorta SM extract, the antigen recognized by the antibody was identified as a decorin precursor. Using a crude decorin preparation from this tissue MM1 reacted strongly with the 33-kDa polypeptide and this pattern did not change after chondroitinase ABC treatment. In vitro, decorin immunoreactivity was found in secreted grainy material produced by confluent arterial SM cells, although lesser amounts were also seen in venous SM cells. Western blotting of extracts from these cultures showed the presence of the 33-kDa band but not of the high-molecular-weight components, except for the 100-kDa monomer. The 100/33-kDa combination was more abundant in arterial SM cells than in the venous counterpart. In the early phase of neointima formation, induced by endothelial injury of the carotid artery or vein-to-artery transposition, the decorin precursor was not expressed, but it was up-regulated in the SM cells of the media underlying the neointima in both models. Collectively, these data suggest a different processing/utilization of the 100-kDa monomer of proteoglycan decorin in arterial and venous SM cells, which is abolished after vein injury. [ABSTRACT FROM AUTHOR]

: ObjectivesWe investigated the potential association between −514 C→T polymorphism in the promoter of the hepatic lipase gene (LIPC) and the prevalence of inflammatory cells in the plaque of patients with severe carotid artery stenosis.: BackgroundThis common LIPC polymorphism has been related to the presence of an atherogenic lipoprotein pattern.: MethodsWe studied 68 consecutive patients undergoing carotid endarterectomy. The LIPC genotype was determined by polymerase chain reaction. Endarterectomy specimens were examined by immunocytochemistry using monoclonal antibodies for smooth muscle cells, macrophages, or lymphocytes.: ResultsIn 50 of 68 patients who had evidence of previous ipsilateral ischemic events, 36 (72%) were carriers of the CC genotype, whereas only 14 (28%) were carriers of the CT/TT genotype (p = 0.002). Among the 18 patients without evidence of events, the two genotypes were equally distributed (9 vs. 9). The low-density lipoprotein (LDL) particles were denser in CC than in CT/TT genotype carriers (flotation rate: 0.315 ± 0.025 vs. 0.356 ± 0.019, p < 0.0005). The CC genotype was associated with an abundance of macrophages (6.7 ± 3.5 vs. 2.1 ± 2.1 cells/area unit in the CT/TT group, p < 0.0005) and a reduced number of smooth muscle cells (6.9 ± 6.2 vs. 14.5 ± 6.4 in the CT/TT group, p < 0.0005) in the plaque. An inverse relationship was found between LDL buoyancy and the number of macrophages in the plaque (r = −0.639, p < 0.0005).: ConclusionsWe provide evidence, for the first time, that LIPC promoter −514 C→T polymorphism, by modulating LDL density, significantly affects the number of macrophages in the plaque and possibly affects the occurrence of cerebrovascular events in patients with carotid artery stenosis. [Copyright &y& Elsevier]

Background: We investigated the effect of nifedipine, AT-1 and ET-1 receptor blockade on arterial smooth muscle cell phenotypes and collagen deposition in TGRen2 transgenic rat (TGR). Methods: Four-week-old TGR were blood pressure (BP)-matched and allocated to receive a placebo (n=8), the calcium antagonist nifedipine (n=6), the AT-1 specific receptor antagonist irbesartan (n=6), the ETA/ETB antagonist bosentan (n=6) or the ETA-selective antagonist BMS-182874 (n=5). Sprague–Dawley normotensive rats served as controls (n=6). After 4 weeks of treatment animals were euthanized and the left ventricle (LV) and the structural changes in intracardiac arterioles and aorta were assessed histomorphometrically. Smooth muscle cell phenotypes and fibrillar collagen content of the aortic wall were evaluated by immunostaining, using differentiation markers—specific antibodies and Syrius red staining, respectively. The changes in ETA and ETB receptor density were also assessed with quantitative autoradiography. Results: Compared to placebo, only irbesartan lowered BP (P<0.001) and prevented LV and small resistance artery hypertrophy. The aorta of placebo-treated TGR showed an increase in foetal-type smooth muscle cell content and fibrillar collagen staining, compared to controls. These changes were blunted by irbesartan, which increased ETA receptors in the arterial wall, enhanced by BMS-182874 and unaffected by bosentan. Nifedipine also blunted both the VSMC and collagen changes despite having no effect on BP and ETA receptors. Conclusions: In TGRen2, vascular hypertrophy entails both smooth muscle cell phenotypic modulation and collagen deposition. These alterations do not follow closely the BP changes and seem to imply the dihydropyridine-sensitive calcium channels. [Copyright &y& Elsevier]

Summary. May–Hegglin anomaly (MHA), Sebastian syndrome (SBS) and Fechtner syndrome (FTNS) are autosomal-dominant macrothrombocytopenias with Döhle-like leucocyte inclusions. These diseases are due to mutations of the MHY9 gene, encoding the heavy chain of non-muscle myosin IIA (NMMHC-A). We investigated the NMMHC-A localization in blood cells from eight MHA, SBS or FTNS patients with known MYH9 mutations. All the patients showed an altered localization of NMMHC-A in granulocytes and platelets, suggesting that Döhle-like bodies are due to the aggregation of NMMHC-A in the cytoplasm. Therefore, immunocytochemistry for NMMHC-A is a simple and sensitive method to detect pathological phenotypes of granulocytes and platelets in the diagnosis of MYH9 -related disorders. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate whether cerivastatin (BAYw6228), a new potent 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, was able to prevent atherogenesis in heterozygous Watanabe heritable-hyperlipidemic (WHHL) rabbits, a model never tested before using this HMG-CoA reductase inhibitor. The heterozygous WHHL rabbits of our breeding developed mild hypercholesterolemia along with focal atherosclerotic lesions in the thoracic aorta. A 9-week treatment with cerivastatin at doses comparable to those used in humans (50 μg/kg/day) reduced serum total cholesterol levels (from 94.4 ± 10.9 to 43.6 ± 10.5 mg/dl, p < 0.005) and prevented aortic lesion development (intima/media ratio: 0.058 ± 0.032 vs 0.946 ± 0.282 in the placebo group, p < 0.0005). Using a panel of monoclonal antibodies specific to macrophages and able to recognize different smooth muscle cell (SMC) phenotypes, we observed that cerivastatin treatment affected the differentiation properties of SMCs and drastically reduced SMC and macrophage accumulation in the intima of the thoracic aorta. These data show that in the presence of moderate atherosclerotic lesions, such as those of heterozygous WHHL rabbits, low doses of cerivastatin exert an antiatherogenic effect.Copyright © 2000 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Two monoclonal antibodies specific for smooth muscle myosin (designated SM-E7 and SM-A9) and one monoclonal anti-(human platelet myosin) antibody (designated NM-G2) have been used to study myosin heavy chain composition of smooth muscle cells in adult and in developing rabbit aorta. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Western blotting experiments revealed that adult aortic muscle consisted of two myosin heavy chains (MCH) of smooth muscle type, named MHC-1 (205 kDa), and MHC-2 (200 kDa). In the fetal/neonatal stage of development, vascular smooth muscle was found to contain only MHC-1 but not MHC-2. Non-muscle myosin heavy chain, which showed the same electrophoretic mobility as the slower migrating MHC, was expressed in an inverse manner with respect to MHC-2, i.e. it was detectable only in the early stages of development. The distinct pattern of smooth and non-muscle myosin isoform expression during development may be related to the different functional properties of smooth muscle cells during vascular myogenesis. [ABSTRACT FROM AUTHOR]