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Hepatocyte growth factor (HGF) is released by stressed human vascular cells and promotes vascular cell repair responses in both autocrine and paracrine ways. Subjects with a low capacity to express HGF in response to systemic stress have an increased cardiovascular risk. Human atherosclerotic plaques with a low content of HGF have a more unstable phenotype. The present study shows that subjects with a low ability to express HGF in response to metabolic stress have an increased risk to suffer myocardial infarction and stroke.

Background: Given that unwanted hair growth (hirsutism, hypertrichosis) can cause major psychological distress, new pharmacological treatment strategies with safe and effective hair growth inhibitors that do not destroy the hair follicle (HF) and its stem cells need to be developed. Objectives: To establish if osteopontin-derived fragments may modulate human hair growth given that human HFs express the multifunctional, immunomodulatory glycoprotein, osteopontin. Methods: Our hypothesis was tested ex vivo and in vivo by using a newly generated, toxicologically well-characterized, modified osteopontin-derived peptide (FOL-005), which binds to the HF. Results: In organ-cultured human HFs and scalp skin, and in human scalp skin xenotransplants onto SCID mice, FOL-005 treatment (60 nmol L−1 to 3 μmol L−1) significantly promoted premature catagen development without reducing the number of keratin 15-positive HF stem cells or showing signs of drug toxicity. Genome-wide DNA microarray, quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry revealed decreased expression of the hair growth promoter, fibroblast growth factor-7 (FGF7) by FOL-005, while cotreatment of HFs with recombinant FGF7 partially abrogated FOL-005-induced catagen promotion. Conclusions: With caveats in mind, our study identifies this osteopontin-derived peptide as an effective, novel inhibitory principle for human hair growth ex vivo and in vivo, which deserves systematic clinical testing in hirsutism and hypertrichosis. What's already known about this topic?. The treatment of unwanted hair growth (hypertrichosis, hirsutism) lacks pharmacological intervention, with only few and often unsatisfactory treatments available. Osteopontin is prominently expressed in human HFs and has been reported to be elevated during catagen in the murine hair cycle. What does this study add?. We tested the effects on hair growth of a novel, osteopontin-derived fragment (FOL-005) ex vivo and in vivo. In human hair follicles, high-dose FOL-005 significantly reduces hair growth both ex vivo and in vivo. What is the translational message?. High-dose FOL-005 may provide a new therapeutic opportunity as a treatment for unwanted hair growth.

Visual Abstract, Highlights • There is strong experimental evidence that IL-5 has a protective role in atherosclerosis but the clinical importance of this remains poorly studied. • In a prospective study involving 696 subjects with a follow-up of close to 17 years we show that baseline plasma levels of IL-5 do not predict risk for coronary events and stroke. • However, subjects with high levels of IL-5 were less likely to have a carotid plaque at the baseline investigation. • Experimental studies using a shear stress-modifying cast to the carotid artery of Apoe−/− mice deficient for IL-5 showed that lack of IL-5 was associated with increased plaque formation at sites of oscillatory blood flow. • The findings are in line with previous experimental observations of an atheroprotective role of IL-5 but do not support the use of IL-5 measurement in cardiovascular risk prediction., Summary Experimental studies have suggested an atheroprotective role of interleukin (IL)-5 through the stimulation of natural immunoglobulin M antibody expression. In the present study we show that there are no associations between baseline levels of IL-5 and risk for development of coronary events or stroke during a 15.7 ± 6.3 years follow-up of 696 subjects randomly sampled from the Malmö Diet and Cancer study. However, presence of a plaque at the carotid bifurcation was associated with lower IL-5 and IL-5 deficiency resulted in increased plaque development at sites of oscillatory blood flow in Apoe−/− mice suggesting a protective role for IL-5 in plaque development.

Background: Acute cardiovascular events arise when the arterial wall fails to repair the damage caused by cardiovascular risk factors. Hepatocyte growth factor (HGF) stimulates growth of vascular cells but has been associated with increased risk of cardiovascular disease (CVD) in epidemiological studies. Here we tested the hypothesis that this association reflects activation of a protective vascular repair response. Methods: Small interfering RNA (siRNA) and recombinant human HGF were used to assess the role of HGF in regulation of human umbilical cord endothelial and coronary artery smooth muscle cell proliferation, migration and viability. Proximity extension assay was used to determine HGF and other biomarkers in 4742 subjects participating in the Malmo Diet and Cancer study and in extracts of 200 human carotid plaques. Findings: HGF promoted migration, proliferation and survival of human endothelial and smooth muscle cells. Human atherosclerotic plaques with a high content of HGF had a more stable phenotype. Cultured vascular cells expressed HGF in response to stress. Plasma HGF correlated with circulating levels of cellular stress biomarkers such as soluble TRAIL receptor-2 and caspase-8. Decreased ability to respond with elevated HGF levels to cellular stress, as assessed by the ratios of HGF to soluble TRAIL receptor-2 and caspase-8, associated with increased risk of cardiovascular events during a follow-up period of 19.4±5.0 years. Interpretations: These findings provide support for a protective role of HGF in CVD and show that subjects with a low ability to express HGF in response to stress have an increased risk of cardiovascular events. Funding Information: The work was supported by the Swedish Society for Medical Research, Emil and Wera Cornell foundation, Hjelt foundation, the Swedish Research Council, the Swedish Foundation for International Cooperation in Research and Higher Education, Crafoord foundation, the Swedish Medical Society, Diabetes foundation, Swedish Heart and Lung Foundation, Diabetes Research and Wellness foundation, Albert Pahlssons foundation, SUS foundations and funds, Lund University Infrastructure grant ”Malmo population-based cohorts” (STYR 2019/2046) and Lund University Diabetes Center (Swedish Research Council - Strategic Research Area Exodiab Dnr 2009-1039 and the Swedish Foundation for Strategic Research Dnr IRC15-006). Declaration of Interests: None. Ethics Approval Statement: All studies were approved by the respective Regional Ethical Review Boards and conducted in accordance with the Helsinki Declaration. All subjects gave written consent.

Placental growth factor (PlGF) is a mitogen for endothelial cells, but it can also act as a proinflammatory cytokine. Because it promotes early stages of plaque formation in experimental models of atherosclerosis and was implicated in epidemiological associations with risk of cardiovascular disease (CVD), PlGF has been attributed a pro-atherogenic role. Here, we investigated whether PlGF has a protective role in CVD and whether elevated PlGF reflects activation of repair processes in response to vascular stress. In a population cohort of 4742 individuals with 20 years of follow-up, high baseline plasma PlGF was associated with increased risk of cardiovascular death, myocardial infarction, and stroke, but these associations were lost or weakened when adjusting for cardiovascular risk factors known to cause vascular stress. Exposure of cultured endothelial cells to high glucose, oxidized low-density lipoprotein (LDL) or an inducer of apoptosis enhanced the release of PlGF. Smooth muscle cells and endothelial cells treated with PlGF small interference RNA demonstrated that autocrine PlGF stimulation plays an important role in vascular repair responses. High expression of PlGF in human carotid plaques removed at surgery was associated with a more stable plaque phenotype and a lower risk of future cardiovascular events. When adjusting associations of PlGF with cardiovascular risk in the population cohort for plasma soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-2, a biomarker of cellular stress, a high PlGF/TRAIL receptor-2 ratio was associated with a lower risk. Our findings provide evidence for a protective role of PlGF in CVD.

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.

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.

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. (Less)

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.

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.

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.

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.

SummaryWhilst innate B1-B cells are atheroprotective, adaptive B2-B cells are considered pro-atherogenic. Different subsets of B regulatory cells (Breg) have been described. In experimental arthritis and lupus-like disease, Breg are contained within the CD21hiCD23hiCD24hi B cell pool. The existence and role of Breg in vascular disease is not known. We sought to investigate the existence, identity and location of Breg 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 CD21hiCD23hiCD24hi B cells are unexpectedly increased in the draining LNs of ApoE-/- mice. Adoptive transfer of LN-derived B2-B cells or purified CD21hiCD23hiCD24hi 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-Breg subset that confers IL-10 mediated protection from neointima formation. This may open the way for immune modulatory approaches in cardiovascular disease.

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.

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 β-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 β-sarcoglycan, and their plaques contained an increase in differentiated smooth muscle cells. ApoE-deficient mice lacking β-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 β-sarcoglycan. Analysis of plasma in β-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 β-sarcoglycan can affect the atherosclerotic process. Furthermore, the results show the effects of β-sarcoglycan deficiency on adipose tissue and lipid metabolism, which may also have contributed to the atherosclerotic plaque reduction.

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 β-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 β-sarcoglycan, and their plaques contained an increase in differentiated smooth muscle cells. ApoE-deficient mice lacking β-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 β-sarcoglycan. Analysis of plasma in β-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 β-sarcoglycan can affect the atherosclerotic process. Furthermore, the results show the effects of β-sarcoglycan deficiency on adipose tissue and lipid metabolism, which may also have contributed to the atherosclerotic plaque reduction.

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.This review covers literature focused on collagen and ECM proteins interacting with collagen, and what their roles may be in plaque development.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.