Positive Smooth Muscle Cells Research Articles

Smooth muscle cell-specific deletion of S100A4 modifies smooth muscle cell fate and inflammation in murine atherosclerotic lesions

Abstract Background S100A4, a calcium-binding protein, is crucial in smooth muscle cell (SMC) phenotypic switch, yet its role in atherosclerotic plaque development and SMC plasticity remains unclear. Our previous experiments using an S100A4 neutralizing antibody approach in ApoE-/- atherosclerotic-prone mice showed reduced atherosclerotic burden but did not establish a direct link between the various S100A4-expressing cells and atherosclerosis. Methods/Results Herein, we show that aortic Oil RedO staining in ApoE-/-; S100A4-/- mice subjected to a 12-week high-cholesterol-diet (HCD) revealed reduced lesion areas compared to ApoE-/- mice (Figure 1, from 21.29% of the total aorta in ApoE-/-; S100A4+/+ mice to 13.27% in ApoE-/-; S100A4-/- mice), highlighting a pivotal role for S100A4 in atherosclerotic plaque development. We then used a lineage tracing ApoE-/- mouse model, in which we induced an SMC-specific deletion of S100A4 (SMC-S100A4Δ/Δ). After 12 weeks of HCD, SMC-S100A4Δ/Δ and control mice (SMC-S100A4wt/wt) were sacrificed, and aortas were processed for en face Oil Red O staining, immunohistochemistry, and single-cell RNA sequencing (scRNA-seq). SMC-specific deletion of S100A4 reduced the necrotic core area (from 29.75% of total plaque area in SMC-S100A4wt/wt to 18.23% in SMC-S100A4Δ/Δ mice) without affecting total atherosclerotic plaque size suggesting a more potent impact on plaque composition. ScRNA-seq analysis from SMC-S100A4Δ/Δ and SMC-S100A4wt/wt mice showed that the inflammatory macrophage-like SMC phenotype was highly suppressed in the plaque and associated with increased SMC repair phenotypes as well as a global reduction of myeloid inflammatory cells. SMCs also retained specific contractile SMC markers, namely Acta2 and Myh11 (fold change of 2.285 and 2.492, respectively) hinting towards a more stable fibrous cap. Immunofluorescence staining on aortic roots plaque (Figure 2) confirmed those results with an increase of SMCs (from 11.02% to 16.18%) as well as a-smooth muscle actin (a-SMA, Acta2)-positive SMCs (from 3.25% to 5.1%) in SMC-S100A4 Δ/Δ mice. Furthermore, the Gene Set Enrichment Analysis (GSEA) of scRNAseq data revealed that, in SMC-S100A4Δ/Δ mice, genes related to mitochondrial metabolism and oxidative respiration were highly downregulated in all SMC clusters. We investigated SMC mitochondrial fitness in vitro and showed that S100A4-/- SMCs have an increased basal oxygen consumption rate and an increased proton leak (fold change of 1.37 and 1.96 respectively) suggestive of differences in cellular metabolic adaptability. Conclusion Our research emphasizes the significant role of S100A4 in plaque development, demonstrating how SMC-derived S100A4 knockdown affects plaque evolution, reducing inflammation, and promoting stability likely through influencing SMC phenotypic switch through metabolic pathways.

The effect of fibroblast growth factor 2 on neovascular vessels depends on the stage of angiogenesis.

ObjectiveThe exact relationship between fibroblast growth factor 2 (FGF2) and choroidal neovascularization (CNV) remains unclear. In this study, using optical coherence tomography angiography (OCTA) and FGF2-tg mice which are transgenic mice with a rhodopsin promoter/FGF2 gene fusion, we aimed to investigate the dynamics of FGF2's role in angiogenesis over time. MethodsWe developed laser-induced CNV models of FGF2-tg and wild-type (WT) mice and then separated them into two groups using different laser photocoagulation (PC) conditions. The first group received 3 intense PC shots (1st PC) altogether (one-time PC group), while the other group received 3 intense PC shots (1st PC) followed by 6 additional weak PC shots (2nd PC) on the 7th day after 1st PC (two-times PC group). ResultsUsing OCTA to observe vessel changes within the same individual over time, there was no difference in the timing of vessel transition from the CNV development phase to the CNV regression phase between FGF2-tg and WT mice in the one-time PC group. In contrast, the neovascular vessels in the two-times PC group of FGF2-tg mice were maintained at least 28 days post-2nd PC without regression. In addition, mature vessels surrounded by PDGFRβ positive pericytes and α-SMA positive smooth muscle cells were observed. Real-time qPCR showed a substantial increase in apelin mRNA expression in the one-time PC group of FGF2-tg, rather than VEGF-A (p<0.05, n=5 or 6). Moreover, the expression levels of PDGFRβ, apelin, and Ang1 were significantly higher in FGF2-tg mice of two-times PC group than in WT mice (p<0.05, n=5 or 6). ConclusionsFGF2 not only promotes neovascularization via the apelin/APJ system, which is independent of VEGF signaling pathway, but also helps maintain and stabilize pre-existing neovascular vessels by stimulating PDGFRβ and Ang1. The effect of FGF2 on the neovascular vessels depends on the stage of angiogenesis.

Smooth muscle cell-specific deletion of S100A4 modifies smooth muscle cell fate and inflammation in murine atherosclerotic lesions

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation (SNSF) S100A4, a calcium-binding protein, is crucial in smooth muscle cell (SMC) phenotypic switch, yet its role in atherosclerotic plaque development and SMC plasticity remains unclear. Our previous experiments using an S100A4 neutralizing antibody approach in ApoE-/- atherosclerotic-prone mice showed reduced atherosclerotic burden but did not establish a direct link between the various S100A4-expressing cells and atherosclerosis. Herein, we show that aortic Oil RedO staining in ApoE-/-; S100A4-/- mice subjected to a 12-week high-cholesterol-diet (HCD) revealed reduced lesion areas compared to ApoE-/- mice (Figure 1, from 21.29% of the total aorta in ApoE-/-; S100A4+/+ mice to 13.27% in ApoE-/-; S100A4-/- mice), highlighting a pivotal role for S100A4 in atherosclerotic plaque development. We then used a lineage tracing ApoE-/- mouse model, in which we induced an SMC-specific deletion of S100A4 (SMC-S100A4Δ/Δ). After 12 weeks of HCD, SMC-S100A4Δ/Δ and control mice (SMC-S100A4wt/wt) were sacrificed, and aortas were processed for en face Oil Red O staining, immunohistochemistry, and single-cell RNA sequencing (scRNA-seq). SMC-specific deletion of S100A4 reduced the necrotic core area (from 29.75% of total plaque area in SMC-S100A4wt/wt to 18.23% in SMC-S100A4Δ/Δ mice) without affecting total atherosclerotic plaque size suggesting a more potent impact on plaque composition. ScRNA-seq analysis from SMC-S100A4Δ/Δ and SMC-S100A4wt/wt mice showed that the inflammatory macrophage-like SMC phenotype was highly suppressed in the plaque and associated with increased SMC repair phenotypes as well as a global reduction of myeloid inflammatory cells. SMCs also retained specific contractile SMC markers, namely Acta2 and Myh11 (fold change of 2.285 and 2.492, respectively) hinting towards a more stable fibrous cap. Immunofluorescence staining on aortic roots plaque (Figure 2) confirmed those results with an increase of SMCs (from 11.02% to 16.18%) as well as a-smooth muscle actin (a-SMA, Acta2)-positive SMCs (from 3.25% to 5.1%) in SMC-S100A4 Δ/Δ mice. Furthermore, the Gene Set Enrichment Analysis (GSEA) of scRNAseq data revealed that, in SMC-S100A4Δ/Δ mice, genes related to mitochondrial metabolism and oxidative respiration were highly downregulated in all SMC clusters. We investigated SMC mitochondrial fitness in vitro and showed that S100A4-/- SMCs have an increased basal oxygen consumption rate and an increased proton leak (fold change of 1.37 and 1.96 respectively) suggestive of differences in cellular metabolic adaptability. In conclusion, our research emphasizes the significant role of S100A4 in plaque development, demonstrating how SMC-derived S100A4 knockdown affects plaque evolution, reducing inflammation, and promoting stability likely through influencing SMC phenotypic switch through metabolic pathways.

Plasma polymerized nanoparticles are a safe platform for direct delivery of growth factor therapy to the injured heart.

Introduction: Heart failure due to myocardial infarction is a progressive and debilitating condition, affecting millions worldwide. Novel treatment strategies are desperately needed to minimise cardiomyocyte damage after myocardial infarction and to promote repair and regeneration of the injured heart muscle. Plasma polymerized nanoparticles (PPN) are a new class of nanocarriers which allow for a facile, one-step functionalization with molecular cargo. Methods: Here, we conjugated platelet-derived growth factor AB (PDGF-AB) to PPN, engineering a stable nano-formulation, as demonstrated by optimal hydrodynamic parameters, including hydrodynamic size distribution, polydisperse index (PDI) and zeta potential, and further demonstrated safety and bioactivity in vitro and in vivo. We delivered PPN-PDGF-AB to human cardiac cells and directly to the injured rodent heart. Results: We found no evidence of cytotoxicity after delivery of PPN or PPN-PDGFAB to cardiomyocytes in vitro, as determined through viability and mitochondrial membrane potential assays. We then measured contractile amplitude of human stem cell derived cardiomyocytes and found no detrimental effect of PPN on cardiomyocyte contractility. We also confirmed that PDGF-AB remains functional when bound to PPN, with PDGF receptor alpha positive human coronary artery vascular smooth muscle cells and cardiac fibroblasts demonstrating migratory and phenotypic responses to PPN-PDGF-AB in the same manner as to unbound PDGF-AB. In our rodent model of PPN-PDGF-AB treatment after myocardial infarction, we found a modest improvement in cardiac function in PPN-PDGF-AB treated hearts compared to those treated with PPN, although this was not accompanied by changes in infarct scar size, scar composition, or border zone vessel density. Discussion: These results demonstrate safety and feasibility of the PPN platform for delivery of therapeutics directly to the myocardium. Future work will optimize PPN-PDGF-AB formulations for systemic delivery, including effective dosage and timing to enhance efficacy and bioavailability, and ultimately improve the therapeutic benefits of PDGF-AB in the treatment of heart failure cause by myocardial infarction.

Anatomical and Histological Study of the Upper Tarsus in Asian.

The aim of this study was to observe and analyze the anatomical and histological characteristics of the upper tarsus in Asian. A total of 15 Asians (14 adults, 1 child) were used. The sagittal sections with thickness of 3μm in the middle of the upper eyelid were prepared and stained with hematoxylin-eosin, Masson trichrome and anti-smooth muscle actin antibody staining, and then were observed and photographed under light microscopy. On the sagittal section, the upper end of the tarsus was connected with the Müller muscle and the part of the posterior layer of the levator aponeurosis; the lower end was the inflated part of the palpebral margin; the superficial surface was connected with the pretarsal fascia; and the deep surface was connected with Müller muscle aponeurosis and palpebral conjunctiva. Histologically, the tarsus was similar to the structure between dense fibrous connective tissue and cartilage, and its main structures include meibomian glands and its ducts, accessory lacrimal glands, glands of Moll, glands of Zeis, eyelash hair follicles, Riolan muscle, blood vessels, and collagen fibers. Through the observation of the tarsus of a child, compared with the adult specimen, the meibomian gland tissue was more abundant; the collagen fibers density was significantly lower and arranged orderly; the blood vessel density was significantly higher; α-SMA positive smooth muscle cells could be seen in the upper end of the tarsus and its superficial and deep surfaces. In this study, the internal and adjacent anatomical structures of the upper tarsus in Asians were observed under a microscope, and according to the histological characteristics, the tarsus was divided into 3 parts and 3 types. Through the preliminary analysis, the tarsus was likely to show different histological characteristics in different individuals and age groups of Asians. There was a certain correlation between the degree of meibomian gland atrophy, the degree of collagen fibrosis and the density of blood vessel in the tarsus.

The Role of CD 34 Hematopoietic Progenitor Cells, Macrophages, and Smooth Muscle Cells in Human Coronary Artery Atherogenesis

BACKGROUND: Atherosclerosis is a widespread and devastating disease and one of the leading causes of death worldwide. So much is there to understand about atherosclerosis. And although a lot is already discovered, yet most of the studies are performed in cell cultures and animal models. Recent technologies for genetic engineering and imaging are mainly performed on animal models, with few studies in human tissues. A better understanding of their role is required.&#x0D; AIM: We aim to study the expression of CD 34 hematopoietic progenitor stem cell, CD 68 macrophages, and smooth muscle actin (SMA)-positive smooth muscle cells (SMCs) in the human coronary arteries and correlate their differential expression with the atherosclerosis progression.&#x0D; RESULTS: CD 68 and CD 34 expression increase as the atherosclerotic process proceeds from early atheroma to advanced atheroma and start to decrease as the process proceeds to fibroatheroma with a significant p &lt; 0.001. Conversely, SMA expression decreases as the atherosclerotic process progresses with a significant p &lt; 0.001.&#x0D; CONCLUSION: CD34 progenitor cells in conjunction with CD 68 macrophages have a major role in the development of atherosclerosis, whereas the SMCs are minimal in the early stages and reach their maximal levels during the stage of fibroatheroma.

Distinct Expression of Nonmuscle Myosin IIB in Pulmonary Arteries of Patients With Aortic Stenosis vs Insufficiency Undergoing a Ross Procedure

BackgroundClinical studies have revealed a greater risk of pulmonary autograft dilation after the Ross procedure in patients with preoperative aortic insufficiency (AI). The present study examined whether the morphologic, biomechanical, and cellular properties of the pulmonary artery (PA) from patients with AI were phenotypically different compared with patients diagnosed with aortic stenosis (AS). MethodsPA segments were harvested from patients undergoing the Ross procedure for AS (n = 16) and AI (n = 6). Preoperative aortic annulus was significantly larger (P < 0.05) in patients with AI (28.5 ± 1.8 mm) vs AS (22.8 ± 1.2 mm). Morphologic, biomechanical, and cellular phenotypes of the PA were analyzed. ResultsCollagen and elastin content in the media of the PA wall were similar in patients with AS and AI. Elastic modulus and energy loss of the PA were not significantly different between the groups. In the media of the PA, expression of a panel of vascular smooth muscle cell–specific proteins were similar in patients with AS and AI. In contrast, nonmuscle myosin IIB protein levels in the PA of AS patients were significantly higher compared with AI patients, and immunofluorescence identified staining in α-smooth muscle actin–positive vascular smooth muscle cells. ConclusionsDespite similar morphological and biomechanical properties, the disparate expression of nonmuscle myosin IIB protein distinguishes the PA of patients with AI from patients with AS. The biological role in vascular smooth muscle cells and the potential contribution of nonmuscle myosin IIB to pulmonary autograft dilation in a subset of AI patients after the Ross procedure remain to be determined.

M2 macrophages promote vasculogenesis during retinal neovascularization by regulating bone marrow-derived cells via SDF-1/VEGF.

Macrophages promote vasculogenesis during retinal neovascularization (RNV) by increasing the recruitment and differentiation of bone marrow-derived cells (BMCs). Different subtypes of macrophages (M1 and M2 macrophages) are associated with RNV. However, the mechanism underlying the regulation of BMCs by different macrophage subtypes during RNV remains unclear. In the present study, we investigated the role and mechanism of action of different macrophage subtypes that regulate BMCs during the development of RNV. The retinal avascular area and neovascularization (NV) tuft area in M2 macrophage group in vivo were the largest compared to those in the control phosphate buffer saline (PBS), unpolarized-M0, and M1 macrophage groups. The number of recruited green fluorescent protein (GFP)-positive BMCs and the degree of differentiation of BMCs into CD31-positive endothelial cells (ECs) and alpha-smooth muscle actin (α-SMA)-positive smooth muscle cells (SMCs) were higher in the M2 macrophage group than in the other groups. M2-conditional medium (M2-CM) affected the in vitro migration and activation of bone marrow mesenchymal stem cells (BMSCs, a subset of BMCs) more than M1-CM. The expression of stromal cell-derived factor-1 (SDF-1) and vascular endothelial growth factor (VEGF) in M2 macrophages and BMSCs cultured with M2-CM was also higher than that in M1 macrophages and BMSCs cultured with M1-CM. Migration of BMSCs was reduced after inhibiting the SDF-1 signaling pathway. Our results indicate that M2 macrophages may express significantly higher levels of SDF-1 and VEGF than M1 macrophages, thus regulating the recruitment and differentiation of BMCs and further aggravating vasculogenesis during RNV.

A novel mouse model of coronary stenosis mimicking Kawasaki disease induced by Lactobacillus casei cell wall extract.

Kawasaki disease (KD), a febrile systemic vasculitis in infants associated with coronary aneurysm, is a major cause of cardiac sequelae such as myocardial infarction (MI) and sudden death. These events are caused by coronary stenosis due to intimal proliferation or thrombotic formation; however, histological evaluation is limited to autopsy cases of human KD. We therefore investigated the histological features of coronary artery (CA) stenosis in mice induced by Lactobacillus casei cell wall extract (LCWE). LCWE-induced coronary inflammation gradually progressed in a time-dependent manner and expanded to all layers of the vessel wall over 28 days. In addition, frequent elastin degradation was observed and abundant α-smooth muscle actin (SMA)-positive vascular smooth muscle cells (VSMCs) infiltrated into the intima. Furthermore, most VSMCs were positive for proliferating cell nuclear antigen (PCNA) following staining, suggesting that VSMCs likely exhibited a proliferative phenotype. In conclusion, we show a novel mouse model of coronary stenosis induced by LCWE that is characterized by coronary stenosis with severe coronary vasculitis and elastin degradation. In addition, VSMC proliferation plays an important role in the formation of coronary stenosis. This model is an appropriate model of KD coronary stenosis.

Foxc1 is required for embryonic head vascular smooth muscle differentiation in zebrafish

Vascular smooth muscle of the head derives from neural crest, but developmental mechanisms and early transcriptional drivers of the vSMC lineage are not well characterized. We find that in early development, the transcription factor foxc1b is expressed in mesenchymal cells that associate with the vascular endothelium. Using timelapse imaging, we observe that foxc1b expressing mesenchymal cells differentiate into acta2 expressing vascular mural cells. We show that in zebrafish, while foxc1b is co-expressed in acta2 positive smooth muscle cells that associate with large diameter vessels, it is not co-expressed in capillaries where pdgfrβ positive pericytes are located. In addition to being an early marker of the lineage, foxc1 is essential for vSMC differentiation; we find that foxc1 loss of function mutants have defective vSMC differentiation and that early genetic ablation of foxc1b or acta2 expressing populations blocks vSMC differentiation. Furthermore, foxc1 is expressed upstream of acta2 and is required for acta2 expression in vSMCs. Using RNA-Seq we determine an enriched intersectional gene expression profile using dual expression of foxc1b and acta2 to identify novel vSMC markers. Taken together, our data suggests that foxc1 is a marker of vSMCs and plays a critical functional role in promoting their differentiation.

5-Lipoxagenase deficiency attenuates L-NAME-induced hypertension and vascular remodeling

BackgroundAbnormalities of the L-arginine-nitric oxide pathway induce hypertension. 5-Lipoxygenase (5-LO) is the key enzyme involved in synthesis of leukotrienes (LTs). However, whether nitricoxide synthase dysfunction induces hypertensive vascular remodeling by regulating 5-LO activity and its downstream inflammatory metabolites remains unknown. Methods and resultsSix-week L-NAME treatment significantly induced hypertension and vascular remodeling in both wild-type (WT) and 5-LO–knockout (5-LO–KO) mice, and blood pressure in caudal and carotid arteries was lower in 5-LO–KO than WT mice with L-NAME exposure. On histology, L-NAME induced less media thickness, media-to-lumen ratio, and collagen deposition and fewer Ki-67–positive vascular smooth muscle cells (VSMCs) but more elastin expression in thoracic and mesenteric aortas of 5-LO–KO than L-NAME–treated WT mice. L-NAME significantly increased LT content, including LTB4 and cysteinyl LT (CysLTs), in plasma and neutrophil culture supernatants from WT mice. On immunohistochemistry, L-NAME promoted the colocalization of 5-LO and 5-LO–activating protein on the nuclear envelope of cultured neutrophils, which was accompanied by elevated LT content in culture supernatants. In addition, LTs significantly promoted BrdU incorporation, migration and phenotypic modulation in VSMCs. ConclusionL-NAME may activate the 5-LO/LT pathway in immune cells, such as neutrophils, and promote the products of 5-LO metabolites, including LTB4 and CysLTs, which aggravate vascular remodeling in hypertension. 5-LO deficiency may protect against hypertension and vascular remodeling by reducing levels of 5-LO downstream inflammatory metabolites.

Abstract 16886: Caspase-4/11 Mediates Intima Formation by Increasing Inflammation and Smooth Muscle Cell Migration and Proliferation

Introduction: Intima formation after vessel injury occurs via vascular smooth muscle cell(VSMC) proliferation, migration, dedifferentiation and inflammatory response. Recently, it was demonstrated that caspase-4 (Casp4, human)/caspase11(Casp11, mouse) increases the synthesis and release of inflammatory factors in macrophages, indicating a potential role of Casp4/Casp11 in intima formation. However, to date, the biological role of Casp4/Casp11 in vivo has not been investigated. Therefore, we hypothesize that Casp4/Casp11 deficiency will inhibit intima formation. Method and Results: We measured Casp4 expression and location by immunohistochemistry (IHC) staining in human coronary artery intima lesions from coronary artery disease patients. Surprisingly, we found Casp4 expression was remarkably increased in smooth muscle cells (SMC) and macrophages. We observed similar phenomena of Casp11 expression and location in intima of complete carotid ligation mouse model. These findings imply an important role of Casp4/11 in SMC function. Thus, we performed complete carotid ligation on Casp11 wild type (WT) and knockout (KO) mice.Morphometric analysis demonstrated that Casp11 depletion robustly attenuated intima area and intima/media ratio (I/M) compared with WT mice . Inflammation was quantified by CD45 IHC staining . SMC proliferation was detected by PCNA staining and there was a marked decrease of PCNA positive SMCs. In vitro, Casp4 expression was time- and dose-dependently increased by platelet-derived growth factor(PDGF), angiotensin II(AngII) and TNF-α in human coronary artery SMCs(HCASMCs). Depletion of Casp4 by siRNA decreased expression of cytokines, e.g. IL-1α, IL-1β, IL-6, as well as protein expression of adhesion factors (VCAM-1, ICAM-1, MCP-1) induced by PDGF, AngII or TNF-α. Moreover, HCASMC proliferation stimulated by PDGF was measured by EdU cell proliferation assay and was inhibited by 65% after knockdown of Casp4. Wound scratch assay demonstrated impaired HCASMC migration in Casp4 depleted cells. However, Casp4 has no effects on HCASMCs dedifferentiation. Mechanistically, Casp4 knockdown significantly inhibited NF-κB signaling. Conclusions: Casp4/11 promotes intima formation by increasing SMC proliferation, migration and inflammation through NF κB signaling. Specific inhibition of the Casp4/11 will provide novel therapeutic strategy for vascular remodeling related diseases.

Characteristic of apoptosis of coronary artery cells with atherosclerotic lesions

To study apoptosis of endothelial cells (EC), macrophages (MF) and smooth muscle cells (SMC) in the early atherosclerotic process (prior to plaque formation), elucidate mechanisms of its realization and evaluate effect on progression of atherosclerosis. Histopathological studies were performed on coronary arteries affected by atherosclerosis taken at autopsy of patients with coronary heart disease (n = 63). To detect apoptosis, the TUNEL method was used for calculating the apoptotic index (AI) in paraffin sections. Phenotyping the cells and test for expression of active caspase 3 (AC-3) and apoptosis-inducing factor (AIF) were performed using sections of coronary arteries stained by immunohistochemistry. The study of apoptosis showed a significant (p <0.0001) increase in AI of SMC, EC, MF coronary arteries affected by atherosclerosis compared with unaffected vessels. Progression of the atherosclerotic process led to a decrease of AI of SMC and EC. Statistical analysis revealed moderate feedback between AI of SMC and neointimal thickness in the development of atherosclerotic lesions (r = -0,44, p <0.0001). The amount of the AC-positive SMC and EC at the stage of lipoidosis significantly (p <0.0001) exceeded the number of AC -positive SMC at the liposclerosis stage. Expression of AIF was observed in the nuclei of EC in the newly formed vessels of hyperplastic intima. Early atherosclerotic lesions of the coronary arteries are accompanied by intense apoptosis of SMC, EC and MF. The intensity of apoptosis of SMC and EC decreases with the development of atherosclerosis. Progression of intimal hyperplasia at the early stages of atherosclerosis is associated with reduced apoptosis of smooth muscle cells and enhanced macrophage apoptosis. Apoptosis of SMC and MF is caspase-dependent. Apoptosis of endothelial cells can occur both with the participation of caspases and independently of them (with AIF).

Influence of periostin-positive cell-specific Klf5 deletion on aortic thickening in DOCA-salt hypertensive mice.

Chronic hypertension causes vascular remodeling that is associated with an increase in periostin- (postn) positive cells, including fibroblasts and smooth muscle cells. Krüppel-like factor (KLF) 5, a transcription factor, is also observed in vascular remodeling; however, it is unknown what role KLF5 plays in postn-positive cells during vascular remodeling induced by deoxycorticosterone-acetate (DOCA) salt. We used postn-positive cell-specific Klf5-deficient mice (Klf5PostnKO: Klf5flox/flox; PostnCre/-) and wild-type mice (WT: Klf5flox/flox; Postn-/-). We implanted a DOCA pellet and provided drinking water containing 0.9% NaCl for 8 weeks. The DOCA-salt treatment induced hypertension in both genotypes, as observed by increases in systolic blood pressure. In WT animals, DOCA-salt treatment increased the aortic medial area compared with the non-treated controls. Similarly, Tgfb1 was overexpressed in the aortas of the DOCA-salt treated WT mice compared with the controls. Immunofluorescence staining revealed that fibroblast-specific protein 1 (FSP1)+-α smooth muscle actin (αSMA)+ myofibroblasts exist in the medial area of the WT aortas after DOCA-salt intervention. Importantly, these changes were not observed in the Klf5PostnKO animals. In conclusion, the results of this study suggest that the presence of KLF5 in postn-positive cells contributes to the pathogenesis of aortic thickening induced by DOCA-salt hypertension.

Leukemia Inhibitory Factor (LIF) Inhibition during Mid-Gestation Impairs Trophoblast Invasion and Spiral Artery Remodelling during Pregnancy in Mice.

The placenta forms the interface between the maternal and fetal circulation and is critical for the establishment of a healthy pregnancy. Trophoblast cell proliferation, migration and invasion into the endometrium are fundamental events in the initiation of placentation. Leukemia inhibitory factor (LIF) has been shown to promote trophoblast invasion in vitro, however its precise role in trophoblast invasion in vivo is unknown. We hypothesized that LIF would be required for normal trophoblast invasion and spiral artery remodeling in mice. Both LIF and its receptor (LIFRα) co-localized with cytokeratin-positive invasive endovascular extravillous trophoblasts (EVT) in mouse implantation sites during mid-gestation. Temporally blocking LIF action during specific periods of placental development via administration of our unique LIFRα antagonist, PEGLA, resulted in abnormal trophoblast invasion and impaired spiral artery remodeling compared to PEG control. PEGLA-treated mouse decidual vessels were characterized by retention of α-smooth muscle actin (αSMA)-positive vascular smooth muscle cells (VSMCs), while PEG control decidual vessels were remodelled by cytokeratin-positive trophoblasts. LIF blockade did not alter F4/80-positive decidual macrophage numbers between treatment groups, but resulted in down-regulation of decidual transcript levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10), which are important immune cell activation factors that promote spiral artery remodeling during pregnancy. Our data suggest that LIF plays an important role in trophoblast invasion in vivo and may facilitate trophoblast-decidual-immune cell cross talk to enable adequate spiral artery remodeling.

Localization of P2X receptor subtypes 2, 3 and 7 in human urinary bladder.

BackgroundVoiding dysfunctions are a common problem that has a severe negative impact on the quality of life. Today there is a need for new drug targets for these conditions. The role of ATP receptors in bladder physiology has been studied for some time, primarily in animal models. The aim of this work is to investigate the localization of the ATP receptors P2X2, P2X3 and P2X7 and their colocalization with vimentin and actin in the human urinary bladder.MethodsImmunohistochemical analysis was conducted on full-thickness bladder tissues from fundus and trigonum collected from 15 patients undergoing open radical cystectomy due to chronic cystitis, bladder cancer or locally advanced prostate cancer. Colocalization analyses were performed between the three different P2X subtypes and the structural proteins vimentin and actin. Specimens were examined using epifluorescence microscopy and correlation coefficients were calculated for each costaining as well as the mean distance from the laminin positive basal side of the urothelium to the vimentin positive cells located in the suburothelium.ResultsP2X2 was expressed in vimentin positive cells located in the suburothelium. Less distinct labelling of P2X2 was also observed in actin positive smooth muscle cells and in the urothelium. P2X3 was expressed in vimentin positive cells surrounding the smooth muscle, and in vimentin positive cells located in the suburothelium. Weaker P2X3 labelling was seen in the urothelium. P2X7 was expressed in the smooth muscle cells and the urothelium. In the suburothelium, cells double positive for P2X2 and vimentin where located closer to the urothelium while cells double positive for P2X3 and vimentin where located further from the urothelium.ConclusionThe results from this study demonstrate that there is a significant difference in the expression of the purinergic P2X2, P2X3 and P2X7 receptors in the different histological layers of the human urinary bladder.

Covering the Cover

Covering the Cover

P0250 : Effect of irreversible electroporation (IRE) on the porcine liver: comparison with radiofrequency ablation

Background and Aims: Irreversible electroporation (IRE) is a promising, non-thermal ablation therapy for hepatocellular carcinoma, which, unlike radiofrequency thermal ablation (RFA), induces apoptosis through irreversible formation of nanoscale pores in the cell membrane with delivery of high-voltage electric current and is considered to improve safety in the vicinity of heat-sensitive blood vessel and bile duct. The aim of the present study is to clarify the acute pathological effect of IRE on the liver and also to compare with effect of RFA. Methods: Three pigs underwent ultrasound-guided IRE and RFA of normal liver under general anesthesia and at laparotomy. A total of 9 IRE ablated areas and 3 RFA ablated areas were created. We examined histopathologic findings of ablated areas at 4 hours after therapy with immunohistochemical methods. The pathologic findings according to IRE ablation conditions were compared with findings of RFA. Results: In IRE zones, various degree of congestion and hemorrhage, apoptotic change of hepatocytes, defluxion of portal endothelial cells and cholangitis were observed in hepatic lobules. With the increase in the total pulse number of IRE delivered, these pathological findings became remarkable. NADPH-diaphorase enzyme histochemistry performed in IRE zones demonstrated the stainability is very weak in the hepatic lobule, which reflecting defluxion of hepatocytes and reduced activity of viable hepatocytes in acute phase, while the stainability is lost in the central portion of RFA. Silver impregnation stain and collagen type 4 immunohistochemistry showed that reticular fibers and collagen fibers around the sinusoid remain well-preserved in IRE zones, on the other hand, those in RFA zones are very poorly stained. The vessels in IRE zones demonstrated a relatively intact configuration of positive smooth muscle cells for Caldesmon staining and endothelial cells for Factor 8 staining, with the minimal endothelial damage, while the vessels in RFA zones demonstrated a negative or weakened reaction. Bile duct epithelial cells showed a positive reaction for cytokeratin 7 staining in IRE zones with desquamated cells, while an entirely negative reaction in RFA zones, revealing protein denaturation. Conclusions: Unlike RFA, IRE induces apoptosis of hepatocytes, endothelium of the blood vessels and mucosal membrane of the bile ducts, whereas preservation of sinusoidal framework and vascular structure may have the potential for liver regeneration.

Propranolol targets the contractility of infantile haemangioma‐derived pericytes

Propranolol, a β-adrenergic receptor (AR) antagonist, is an effective treatment for endangering infantile haemangioma (IH). Dramatic fading of cutaneous colour is often seen a short time after initiating propranolol therapy, with accelerated regression of IH blood vessels discerned after weeks to months. To assess a possible role for haemangioma-derived pericytes (HemPericytes) isolated from proliferating and involuting phase tumours in apparent propranolol-induced vasoconstriction. HemPericytes were assayed for contractility on a deformable silicone substrate: propranolol (10 μmol L(-1)) restored basal contractile levels in HemPericytes that were relaxed with the AR agonist epinephrine. Small interfering RNA knockdown of β2-AR blunted this response. HemPericytes and haemangioma-derived endothelial cells were co-implanted subcutaneously in nude mice to form blood vessels; at day 7 after injection, mice were randomized into vehicle and propranolol-treated groups. HemPericytes expressed high levels of β2-AR mRNA compared with positive control bladder smooth muscle cells. In addition, β2-AR mRNA levels were relatively high in IH specimens (n = 15) compared with β1-AR, β3-AR and α1b-AR. Normal human retinal and placental pericytes were not affected by epinephrine or propranolol in this assay. Propranolol (10 μmol L(-1)) inhibited the proliferation of HemPericytes in vitro, as well as normal pericytes, indicating a nonselective effect in this assay. Contrast-enhanced microultrasonography of the implants after 7 days of treatment showed significantly decreased vascular volume in propranolol-treated animals, but no reduction in vehicle-treated animals. These findings suggest that the mechanism of propranolol's effect on proliferating IH involves increased pericytic contractility.

Tu1651 Indian Hedgehog Is Required for Intestinal Adenoma Formation

Introduction: Activating mutations in the Hedgehog pathway are found in basal cell carcinomas and medulloblastomas. The role of Hedgehog signaling in intestinal tumorigenesis has not yet been clarified. Hedgehog is expressed by differentiated enterocytes and signals in a paracrine manner from the epithelium to the mesenchyme. Hedgehog controls mesenchymal factors such as Bone Morphogenetic Proteins and Activins which negatively regulate the proliferation of precursor cells. Since Indian Hedgehog (Ihh) is the major Hedgehog expressed in the intestinal epithelium we decided to study the potential role of Ihh signaling in intestinal adenoma formation.Methods: In order to study the effect of loss of Ihh signaling in sporadic tumorigenesis we conditionally deleted Ihh in adult mice in the intestinal epithelium using the Cyp1a1Cre promoter. These compound mice were crossed with conditional mutant Apc mice to generate Cyp1a1Cre-Ihh-Apc mice. At 4-7 weeks of age mice were intraperitoneally injected with 80 mg/kg β-naphthoflavone for 5 days. Cyp1a1Cre-IhhApc littermates served as controls. Four months after recombination, we sacrificed and analyzed the mice (n=15). Results: Ihh expression was upregulated in polyps of Cyp1a1CreIhh-Apc mice as assessed by in situ hybridization. Deletion of Ihh resulted in a marked reduction of 90.3% in the number of polyps (17.2 ± 13.65 polyps per mouse vs. 1.7 ± 1.5, P < 0.01). The average polyp size did not differ between groups. Neither did the number of proliferating or apoptotic cells in the polyps, as assessed by immunostaining for BrdU and cleaved caspase 3. Hh responsive cells in the adenomawere restricted to themesenchyme. In those adenomas that did develop in Ihh mutant mice, we observed a remarkable loss of α-sma/desmin double positive smooth muscle cells. COX2 is expressed early in adenoma to carcinoma transition and believed to play an important role in adenoma formation. Stromal cells are known to be a source of COX2. In accordance with this, examination of Cox2 expression by Q-PCR and immunostaining showed up regulation in polyps ofCyp1a1Cre-Ihh -Apc mice in compared to normal intestinal epithelium (P < 0.01, resp. P < 0.05). In contrast, Cox2 expression in the polyps of Cyp1a1Cre-Ihh-Apc mice was not significantly up regulated. Conclusion: Surprisingly, in contrast to its role as an anti-proliferative signal in the normal epithelium, Ihh acts as a tumor promoter. Our data show that Ihh signaling is increased during intestinal adenoma formation and as in the normal intestine Ihh signaling is exclusively from the epithelium to the mesenchyme. Loss of Ihh signaling is associated with major changes in the composition of the adenoma mesenchyme and decreased Cox2 expression, which significantly impairs adenoma formation.