Effect of acidosis on adipose-derived stem cell impairment and gene expression.

Based on disappointing results of stem cell-based application in clinical trials for patients with critical limb ischemia, we hypothesized that the acidic environment might be the key factor limiting cell survival and function. In the present study, we used microdialysis to determine presence of acidosis and metabolic imbalance in critical ischemia. Moreover, we explored the effect of extracellular acidosis on adipose-derived stem cells (ADSCs) at molecular and transcriptional level. Our data demonstrate that low pH negatively regulates cell proliferation and survival, also, it results in cell cycle arrest, mitochondrial dynamics disorder, DNA damage as well as the impairment of proangiogenic function in a pH-dependent manner. Further transcriptome profiling identified the pivotal signaling pathways and hub genes in response to acidosis. Collectively, these findings provide strong evidences for a critical role of acidosis in ADSCs impairment with ischemic condition and suggest treatments focus on tissue pH balance and acidosis-mediated hub genes may have therapeutic potential in stem cell-based application.

Cartilage Oligomeric Matrix Protein Associates With a Vulnerable Plaque Phenotype in Human Atherosclerotic Plaques.

Background and Purpose- Extracellular matrix proteins are important in atherosclerotic disease by influencing plaque stability and cellular behavior but also by regulating inflammation. COMP (cartilage oligomeric matrix protein) is present in healthy human arteries and expressed by smooth muscle cells. A recent study showed that transplantation of COMP-deficient bone marrow to apoE-/- mice increased atherosclerotic plaque formation, indicating a role for COMP also in bone marrow-derived cells. Despite the evidence of a role for COMP in murine atherosclerosis, knowledge is lacking about the role of COMP in human atherosclerotic disease. Methods- In the present study, we investigated if COMP was associated with a stable or a vulnerable human atherosclerotic plaque phenotype by analyzing 211 carotid plaques for COMP expression using immunohistochemistry. Results- Plaque area that stained positive for COMP was significantly larger in atherosclerotic plaques associated with symptoms (n=110) compared with asymptomatic plaques (n=101; 9.7% [4.7-14.3] versus 5.6% [2.8-9.8]; P=0.0002). COMP was positively associated with plaque lipids (r=0.32; P=0.000002) and CD68 cells (r=0.15; P=0.036) but was negatively associated with collagen (r=-0.16; P=0.024), elastin (r=-0.14; P=0.041), and smooth muscle cells (r=-0.25; P=0.0002). COMP was positively associated with CD163 (r=0.37; P=0.00000006), a scavenger receptor for hemoglobin/haptoglobin and a marker of Mhem macrophages, and with intraplaque hemorrhage, measured as glycophorin A staining (r=0.28; P=0.00006). Conclusions- The present study shows that COMP is associated to symptomatic carotid atherosclerosis, CD163-expressing cells, and a vulnerable atherosclerotic plaque phenotype in humans.

Osteopontin protects against lung injury caused by extracellular histones.

Extracellular histones are present in the airways because of cell death occurring during inflammation. They promote inflammation and cause tissue damage due to their cationic nature. The anionic phosphoglycoprotein osteopontin (OPN) is expressed at high levels during airway inflammation and has been ascribed both pro- and anti-inflammatory roles. In this study, it was hypothesized that OPN may neutralize the harmful activities of extracellular histones at the airway mucosal surface. In a model of histone-induced acute lung injury, OPN-/- mice showed increased inflammation and tissue injury, and succumbed within 24 h, whereas wild-type mice showed lower degrees of inflammation and no mortality. In lipopolysaccharide-induced acute lung injury, wild-type mice showed less inflammation and tissue injury than OPN-/- mice. In bronchoalveolar lavage fluid from ARDS patients, high levels of OPN and also histone-OPN complexes were detected. In addition, OPN bound to histones with high affinity in vitro, resulting in less cytotoxicity and reduced formation of tissue-damaging neutrophil extracellular traps (NETs). The interaction between OPN and histones was dependent on posttranslational modification of OPN, i.e., phosphorylation. The findings demonstrate a novel role for OPN, modulating the pro-inflammatory and cytotoxic properties of free histones.

Osteopontin protects against pneumococcal infection in a murine model of allergic airway inflammation.

BACKGROUND: In atopic asthma, chronic Th2-biased inflammation is associated with an increased risk of pneumococcal infection. The anionic phosphoglycoprotein osteopontin (OPN) is highly expressed in asthma and has been ascribed several roles during inflammation. This study aimed to investigate whether OPN affects inflammation and vulnerability to pneumococcal infection in atopic asthma. METHODS: House dust mite (HDM) extract was used to induce allergic airway inflammation in both wild-type (Spp1+/+ ) and OPN knockout (Spp1-/- ) C57BL/6J mice, and the airway was then infected with Streptococcus pneumoniae. Parameters reflecting inflammation, tissue injury, and bacterial burden were measured. In addition, samples from humans with allergic asthma were analyzed. RESULTS: Both allergen challenge in individuals with allergic asthma and the intranasal instillation of HDM in mice resulted in increased OPN levels in bronchoalveolar lavage fluid (BALF). More immune cells (including alveolar macrophages, neutrophils, eosinophils, and lymphocytes) and higher levels of proinflammatory cytokines were found in Spp1-/- mice than in Spp1+/+ mice. Moreover, OPN-deficient mice exhibited increased levels of markers reflecting tissue injury. Upon infection with S. pneumoniae, Spp1+/+ mice with allergic airway inflammation had a significantly lower bacterial burden in both BALF and lung tissue than did Spp1-/- mice. Furthermore, Spp1-/- mice had higher levels of cytokines and immune cells in BALF than did Spp1+/+ mice. CONCLUSION: OPN reduces inflammation, decreases tissue injury, and reduces bacterial loads during concurrent pneumococcal infection and allergic airway inflammation in a murine model. These findings suggest that OPN significantly affects vulnerability to pneumococcal infection in atopic asthma.

Hyperglycemia does not affect tissue repair responses in shear stress-induced atherosclerotic plaques in ApoE-/- mice.

The mechanisms responsible for macrovascular complications in diabetes remain to be fully understood. Recent studies have identified impaired vascular repair as a possible cause of plaque vulnerability in diabetes. This notion is supported by observations of a reduced content of fibrous proteins and smooth muscle cell mitogens in carotid endarterectomy from diabetic patients along with findings of decreased circulating levels of endothelial progenitor cells. In the present study we used a diabetic mouse model to characterize how hyperglycemia affects arterial repair responses. We induced atherosclerotic plaque formation in ApoE-deficient (ApoE-/-) and heterozygous glucokinase knockout ApoE-deficient mice (ApoE-/- GK+/-) mice with a shear stress-modifying cast. There were no differences in cholesterol or triglyceride levels between the ApoE-/- and ApoE-/- GK+/- mice. Hyperglycemia did not affect the size of the formed atherosclerotic plaques, and no effects were seen on activation of cell proliferation, smooth muscle cell content or on the expression and localization of collagen, elastin and several other extracellular matrix proteins. The present study demonstrates that hyperglycemia per se has no significant effects on tissue repair processes in injured mouse carotid arteries, suggesting that other mechanisms are involved in diabetic plaque vulnerability.

Distinctive peri-luminal presence of agrin in murine and human carotid atherosclerotic plaques.

The clinical consequences of arterial atherosclerotic lesions depend, apart from their size, on their composition of cellular and extracellular components. While an intact endothelium at the interface of atherosclerotic plaques towards the blood can prevent its erosion, underlying smooth muscle cells within the plaque can reduce the risk of plaque ruptures, due to the deposition of stabilizing extracellular matrix. Basement membranes underlay and support the function of endothelial cells, and embed smooth muscle cells in the media, the source of most smooth muscle cells within atherosclerotic plaques. In the present study mouse atherosclerotic plaques were comparatively analyzed for the basement membrane components laminin, type IV collagen, perlecan, and agrin. Distinct agrin immunofluorescence was found in the peri-luminal area in mouse carotid atherosclerotic plaques. Agrin was also clearly present in the media, with a significant increase in regions directly associated with plaque tissue. In addition, ten human endarterectomy specimens were investigated for this heparan sulfate proteoglycan. No statistically significant differences in agrin immunofluorescence were noticed between five specimens from symptomatic and five from asymptomatic patients. In all these plaques agrin was present in a distinctive manner in a narrow zone partially or almost completely surrounding the lumen. Additionally it was also present around the small lumina of the CD31-positive neovessels. The presence of agrin at locations with particular importance for the growth and stability of atherosclerotic plaques renders this molecule strategically positioned to influence plaque development and vulnerability.

ß-Sarcoglycan Deficiency Reduces Atherosclerotic Plaque Development in ApoE-Null Mice.

BACKGROUND: Smooth muscle cells are important for atherosclerotic plaque stability. Their proper ability to communicate with the extracellular matrix is crucial for maintaining the correct tissue integrity. In this study, we have investigated the role of ß-sarcoglycan within the matrix-binding dystrophin-glycoprotein complex in the development of atherosclerosis. RESULTS: Atherosclerotic plaque development was significantly reduced in ApoE-deficient mice lacking ß-sarcoglycan, and their plaques contained an increase in differentiated smooth muscle cells. ApoE-deficient mice lacking ß-sarcoglycan showed a reduction in ovarian adipose tissue and adipocyte size, while the total weight of the animals was not significantly different. Western blot analysis of adipose tissues showed a decreased activation of protein kinase B, while that of AMP-activated kinase was increased in mice lacking ß-sarcoglycan. Analysis of plasma in ß-sarcoglycan-deficient mice revealed reduced levels of leptin, adiponectin, insulin, cholesterol, and triglycerides but increased levels of IL-6, IL-17, and TNF-α. CONCLUSIONS: Our results indicate that the dystrophin-glycoprotein complex and ß-sarcoglycan can affect the atherosclerotic process. Furthermore, the results show the effects of ß-sarcoglycan deficiency on adipose tissue and lipid metabolism, which may also have contributed to the atherosclerotic plaque reduction.

ADAMTS-7 is associated with a high-risk plaque phenotype in human atherosclerosis.

Several large-scale genome-wide association studies have identified single-nucleotide polymorphisms in the genomic region of A Disintegrin And Metalloproteinase with ThromboSpondin type 1 repeats (ADAMTS)-7 and associations to coronary artery disease. Experimental studies have provided evidence for a functional role of ADAMTS-7 in both injury-induced vascular neointima formation and development of atherosclerotic lesions. However, whether ADAMTS-7 is associated with a specific plaque phenotype in humans has not been investigated. Carotid plaques (n = 206) from patients with and without cerebrovascular symptoms were analyzed for expression of ADAMTS-7 by immunohistochemistry and correlated to components associated with plaque vulnerability. Plaques from symptomatic patients showed increased levels of ADAMTS-7 compared with lesions from asymptomatic patients. High levels of ADAMTS-7 correlated with high levels of CD68-staining and lipid content, but with low smooth muscle cell and collagen content, which together are characteristics of a vulnerable plaque phenotype. ADAMTS-7 levels above median were associated with increased risk for postoperative cardiovascular events. Our data show that ADAMTS-7 is associated with a vulnerable plaque phenotype in human carotid lesions. These data support previous observations of a potential proatherogenic role of ADAMTS-7.

Osteopontin Affects Insulin Vesicle Localization and Ca2+ Homeostasis in Pancreatic Beta Cells from Female Mice.

Type 2 diabetic patients suffer from insulin resistance and reduced insulin secretion. Osteopontin (OPN), a versatile protein expressed in several tissues throughout the body including the islets of Langerhans, has previously been implicated in the development of insulin resistance. Here we have investigated the role of OPN in insulin secretion using an OPN knock out mouse model (OPN-/-). Ultra-structural analyzes of islets from OPN-/- and WT mice indicated weaker cell-cell connections between the islet cells in the OPN-/- mouse compared to WT. Analysis of the insulin granule distribution in the beta cells showed that although OPN-/- and WT beta cells have the same number of insulin granules OPN-/- beta cells have significantly fewer docked granules. Both OPN-/- and WT islets displayed synchronized Ca2+ oscillations indicative of an intact beta cell communication. OPN-/- islets displayed higher intracellular Ca2+ concentrations when stimulated with 16.7 mM glucose than WT islets and the initial dip upon elevated glucose concentrations (which is associated with Ca2+ uptake into ER) was significantly lower in these islets. Glucose-induced insulin secretion was similar in OPN-/- and WT islets. Likewise, non-fasted blood glucose levels were the same in both groups. In summary, deletion of OPN results in several minor beta-cell defects that can be compensated for in a healthy system.

Treatment with a GnRH receptor agonist, but not the GnRH receptor antagonist degarelix, induces atherosclerotic plaque instability in ApoE(-/-) mice.

Androgen-deprivation therapy (ADT) for prostate cancer has been associated with increased risk for development of cardiovascular events and recent pooled analyses of randomized intervention trials suggest that this primarily is the case for patients with pre-existing cardiovascular disease treated with gonadotropin-releasing hormone receptor (GnRH-R) agonists. In the present study we investigated the effects of the GnRH-R agonist leuprolide and the GnRH-R antagonist degarelix on established atherosclerotic plaques in ApoE(-/-) mice. A shear stress modifier was used to produce both advanced and more stable plaques in the carotid artery. After 4 weeks of ADT, increased areas of necrosis was observed in stable plaques from leuprolide-treated mice (median and IQR plaque necrotic area in control, degarelix and leuprolide-treated mice were 0.6% (IQR 0-3.1), 0.2% (IQR 0-4.4) and 11.0% (IQR 1.0-19.8), respectively). There was also evidence of increased inflammation as assessed by macrophage immunohistochemistry in the plaques from leuprolide-treated mice, but we found no evidence of such changes in plaques from control mice or mice treated with degarelix. Necrosis destabilizes plaques and increases the risk for rupture and development of acute cardiovascular events. Destabilization of pre-existing atherosclerotic plaques could explain the increased cardiovascular risk in prostate cancer patients treated with GnRH-R agonists.

Dystrophin deficiency reduces atherosclerotic plaque development in ApoE-null mice.

Dystrophin of the dystrophin-glycoprotein complex connects the actin cytoskeleton to basement membranes and loss of dystrophin results in Duchenne muscular dystrophy. We have previously shown injury-induced neointima formation of the carotid artery in mice with the mdx mutation (causing dystrophin deficiency) to be increased. To investigate the role of dystrophin in intimal recruitment of smooth muscle cells (SMCs) that maintains plaque stability in atherosclerosis we applied a shear stress-modifying cast around the carotid artery of apolipoprotein E (ApoE)-null mice with and without the mdx mutation. The cast induces formation of atherosclerotic plaques of inflammatory and SMC-rich/fibrous phenotypes in regions of low and oscillatory shear stress, respectively. Unexpectedly, presence of the mdx mutation markedly reduced the development of the inflammatory low shear stress plaques. Further characterization of the low shear stress plaques in ApoE-null mdx mice demonstrated reduced infiltration of CD3(+) T cells, less laminin and a higher SMC content. ApoE-null mdx mice were also found to have a reduced fraction of CD3(+) T cells in the spleen and lower levels of cytokines and monocytes in the circulation. The present study is the first to demonstrate a role for dystrophin in atherosclerosis and unexpectedly shows that this primarily involves immune cells.

IL-22 affects smooth muscle cell phenotype and plaque formation in apolipoprotein E knockout mice.

OBJECTIVE: IL-22 is a recently discovered cytokine that belongs to the family of IL-10 related cytokines. It is produced by activated T-cells and innate lymphoid cells and has been suggested to be involved in tissue repair. As both inflammation and repair play important roles in atherosclerosis we investigated if IL-22 deficiency influences the disease process in Apoe(-/-) mice. METHODS: We generated IL-22(-/-)Apoe(-/-) mice and fed them high-fat-diet for 14 weeks to characterize atherosclerosis development. RESULTS: IL-22(-/-)Apoe(-/-) mice exhibited reduced plaque size both in the aorta (p = 0.0036) and the aortic root compared (p = 0.0012) with Apoe(-/-) controls. Moreover, plaque collagen was reduced in IL-22(-/-)Apoe(-/-) mice (p = 0.02) and this was associated with an increased expression of smooth muscle cell (SMC)-α-actin (p = 0.04) and caldesmon (p = 0.016) in the underlying media. Carotid arteries from IL-22(-/-)Apoe(-/-) mice displayed increased expression of genes associated with a contractile SMC phenotype e.g. α-actin (p = 0.004) and caldesmon (p = 0.03). Arterial SMCs were shown to express the IL-22 receptor and in vitro exposure to IL-22 resulted in a down-regulation of alpha actin and caldesmon gene expression in these cells. CONCLUSION: Our observations demonstrate that IL-22 is involved in plaque formation and suggest that IL-22 released by immune cells is involved in activation of vascular repair by stimulating medial SMC dedifferentiation into a synthetic phenotype. This response contributes to plaque growth by enabling SMC migration into the intima but may also help to stabilize the plaque.

Expression of fibromodulin in carotid atherosclerotic plaques is associated with diabetes and cerebrovascular events.

AIMS: The small leucine-rich proteoglycans fibromodulin and lumican are functionally related extracellular matrix proteins involved in the regulation of collagen fiber formation. Fibromodulin-deficient apolipoprotein E-null mice have decreased vascular retention of lipids and reduced development of atherosclerosis suggesting that fibromodulin may influence the disease process. The aim of the present study was to investigate if fibromodulin and lumican are expressed in human carotid plaques and to determine if their expression is associated with the occurrence of preoperative symptoms and with risk for postoperative cardiovascular events. METHODS AND RESULTS: 153 plaques (51% symptomatic) obtained by carotid endarterectomy were included in this study. Plaque content was analyzed by immunohistochemistry and plaque cytokine content by multiplex technology. Fibromodulin and lumican were widely expressed in plaques and fibromodulin expression was significantly higher in symptomatic plaques. Expression of fibromodulin was significantly higher in plaques obtained from patients with diabetes and a high fibromodulin expression was associated with a higher incidence of post-operative cerebrovascular events, whereas no such associations were seen for lumican. Fibromodulin expression also correlated with plaque lipids and several pro-inflammatory cytokines. In addition, fibromodulin expression correlated with low levels of smooth muscle cells and the anti-inflammatory cytokine IL-10. CONCLUSIONS: These observations support previous experimental findings in mice for a role of fibromodulin in atherosclerosis and provide clinical evidence of the involvement of fibromodulin in the inflammatory processes that characterize atherosclerotic plaque vulnerability. They also suggest that this is of particular importance in diabetes.

B regulatory cells are increased in hypercholesterolaemic mice and protect from lesion development via IL-10.

Whilst innate B1-B cells are atheroprotective, adaptive B2-B cells are considered pro-atherogenic. Different subsets of B regulatory cells (B(reg)) have been described. In experimental arthritis and lupus-like disease, B(reg) are contained within the CD21(hi)CD23(hi)CD24(hi) B cell pool. The existence and role of B(reg) in vascular disease is not known. We sought to investigate the existence, identity and location of B(reg) in vascular disease. The representation of B2-B cell subsets in the spleens and lymph nodes (LNs) of Apolipoprotein E(-/-) (ApoE(-/-)) mice compared to controls was characterised by flow cytometry. Additionally, we utilised a model of neointima formation based on the placement of a perivascular collar around the carotid artery in ApoE(-/-) mice to ascertain whether B cells and B cell subsets confer protection against lesion development. Adoptive transfer of B cells was performed from wild type or genetically modified mice. We showed that CD21(hi)CD23(hi)CD24(hi) B cells are unexpectedly increased in the draining LNs of ApoE(-/-) mice. Adoptive transfer of LN-derived B2-B cells or purified CD21(hi)CD23(hi)CD24(hi) B cells to syngeneic mice reduced lesion size and inflammation without changing serum cholesterol levels. Follicular B2-B cells did not confer protection. IL-10 blockade or transfer of IL10-deficient B cells prevented LN-derived B cell-mediated protection. This is the first identification of a specific LN-derived B2-B(reg) subset that confers IL-10 mediated protection from neointima formation. This may open the way for immune modulatory approaches in cardiovascular disease.

Regulation of smooth muscle dystrophin and synaptopodin 2 expression by actin polymerization and vascular injury.

OBJECTIVE: Actin dynamics in vascular smooth muscle is known to regulate contractile differentiation and may play a role in the pathogenesis of vascular disease. However, the list of genes regulated by actin polymerization in smooth muscle remains incomprehensive. Thus, the objective of this study was to identify actin-regulated genes in smooth muscle and to demonstrate the role of these genes in the regulation of vascular smooth muscle phenotype. APPROACH AND RESULTS: Mouse aortic smooth muscle cells were treated with an actin-stabilizing agent, jasplakinolide, and analyzed by microarrays. Several transcripts were upregulated including both known and previously unknown actin-regulated genes. Dystrophin and synaptopodin 2 were selected for further analysis in models of phenotypic modulation and vascular disease. These genes were highly expressed in differentiated versus synthetic smooth muscle and their expression was promoted by the transcription factors myocardin and myocardin-related transcription factor A. Furthermore, the expression of both synaptopodin 2 and dystrophin was significantly reduced in balloon-injured human arteries. Finally, using a dystrophin mutant mdx mouse and synaptopodin 2 knockdown, we demonstrate that these genes are involved in the regulation of smooth muscle differentiation and function. CONCLUSIONS: This study demonstrates novel genes that are promoted by actin polymerization, that regulate smooth muscle function, and that are deregulated in models of vascular disease. Thus, targeting actin polymerization or the genes controlled in this manner can lead to novel therapeutic options against vascular pathologies that involve phenotypic modulation of smooth muscle cells.

Cartilage oligomeric matrix protein (COMP) in murine brachiocephalic and carotid atherosclerotic lesions.

OBJECTIVE: To investigate the hypothesis that COMP can influence the morphology, stability and size of murine atherosclerotic lesions. METHODS: ApoE- and ApoE/COMP-knockout mice were fed a high-fat diet to develop atherosclerotic plaques at lesion sites of three different types; inflammatory and fibrous plaques induced in the carotid artery by low or oscillatory shear stress, respectively, and spontaneously developing plaques in the brachiocephalic artery. The localization of COMP in the plaques and the effect of COMP deficiency on plaque development were evaluated. RESULTS: COMP immunoreactivity was observed in about half of the investigated plaques from the ApoE null mice, mainly located along the intima-medial border. There were no significant differences in the size of inflammatory and fibrous carotid plaques between the genotypes. Plaques in the brachiocephalic artery from ApoE mice lacking COMP were increased in size with 54%. In these plaques the collagen content was also increased by 48%. There were no differences in relative collagen content in inflammatory and fibrous carotid plaques between genotypes. Polarized light microscopy showed that the increase in total collagen in brachiocephalic plaques was more than proportionally accounted for by an increase in thicker collagen fibrils. CONCLUSION: We have shown that COMP deficiency has a significant impact on atherosclerotic plaque morphology and size. Our data also suggest that an altered collagen metabolism may be an important mechanism in this finding.

Collagen and related extracellular matrix proteins in atherosclerotic plaque development.

PURPOSE OF REVIEW: The structure, composition and turnover of the extracellular matrix (ECM) as well as cell-matrix interactions are crucial in the developing atherosclerotic plaque. There is a need for further insight into specific proteins in the ECM and their functions in the developing plaque, and during the last few years a number of publications have highlighted this very important field of research. These novel findings will be addressed in the present review. RECENT FINDINGS: This review covers literature focused on collagen and ECM proteins interacting with collagen, and what their roles may be in plaque development. SUMMARY: Acute myocardial infarction and stroke are common diseases that cause disability and mortality, and the underlying mechanism is often the rupture of a vulnerable atherosclerotic plaque. The vascular ECM and the tissue repair in the atherosclerotic lesion are important players in plaque progression. Understanding how specific proteins in the ECM interact with cells in the plaque and affect the fate of the plaque can lead to new treatments for cardiovascular disease.