Arterial haptoglobin : expression, regulation and function

Several studies have demonstrated that collagen turnover is important during arterial restructuring. However, the precise mediators involved in collagen turnover are still not known. In order to develop specific inhibitors to prevent arterial restructuring, it is essential to get a better understanding of the molecular pathways involved in collagen turnover. In this thesis, we used a subtraction PCR to identify new candidate proteins that are involved in arterial restructuring and to get a better insight in these pathways. One of the identified proteins using subtraction PCR was the acute phase glycoprotein haptoglobin. Haptoglobin was found to be expressed by adventitial fibroblasts, early after flow changes. In vitro assays demonstrated that haptoglobin is a gelatinase inhibitor and important for cell migration. Furthermore, haptoglobin knockout mice demonstrated delayed arterial restructuring after flow changes. As NO plays an important role in flow-induced arterial restructuring, we investigated whether arterial haptoglobin expression was regulated via NO. Inhibition of NO synthesis by a aspecific NOS-inhibitor resulted in decreased arterial haptoglobin expression. This coincided with decreased arterial IL-6 expression, which is a well described regulator of haptoglobin expression in the liver. IL-6 knockout mice were used to confirm a regulatory role of IL-6 in arterial haptoglobin expression. However, haptoglobin expression was normal in IL-6 knockout mice, pointing to back-up mechanisms for the regulation of haptoglobin expression. Previously, sero-epidemiological studies have demonstrated that arthritis and cancer are characterized by increased serum haptoglobin levels. In this thesis, we demonstrated that local expression of haptoglobin is increased in these two pathological tissues that are characterized by enhanced cell migration and matrix turnover. As haptoglobin is present at high concentrations in the serum, the function of local haptoglobin expression is not completely understood. We therefore investigated whether alterations in haptoglobin glycosylation could explain the necessity of local haptoglobin expression. Haptoglobin expression and glycosylation was studied in balloon dilated rabbit arteries. Balloon dilation increased arterial haptoglobin expression within the first two weeks after injury whereas liver haptoglobin expression remained constant. There were no differences in arterial haptoglobin glycosylation patterns after balloon dilation. However, arterial haptoglobin was differentially glycosylated compared to liver haptoglobin and these arterial haptoglobin glycoforms could be detected in the serum. Previously, alterations in serum haptoglobin glycosylation have been associated with the progression and outcome of different diseases. As arterial haptoglobin has a characteristic glycosylation pattern, we investigated whether serum haptoglobin glycosylation patterns could serve as serum marker for atherosclerotic disease. There were, however, no differences were found in total serum haptoglobin levels or serum haptoglobin glycosylation patterns between patients with atherosclerosis and patients with only risk factors. In addition, no relation was found between total serum haptoglobin levels and glycosylation patterns with intima-media thickness in the carotid artery, which is a surrogate measure for the extent of atherosclerosis. These results indicate that serum haptoglobin glycosylation patterns are not suitable to use as serum marker for atherosclerotic diseas

SARS-CoV-2 infects the human kidney and drives fibrosis in kidney organoids

Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human-induced pluripotent stem-cell-derived kidney organoids with SARS-CoV-2. Single-cell RNA sequencing indicated injury and dedifferentiation of infected cells with activation of profibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in long COVID

Annexin A2 Phosphorylation Mediates Cell Scattering and Branching Morphogenesis via Cofilin Activation▿ †

Dynamic remodeling of the actin cytoskeleton is required for cell spreading, motility, and migration and can be regulated by tyrosine kinase activity. Phosphotyrosine proteomic screening revealed phosphorylation of the lipid-, calcium-, and actin-binding protein annexin A2 (AnxA2) at Tyr23 as a major event preceding ts-v-Src kinase-induced cell scattering. Expression of the phospho-mimicking mutant Y23E-AnxA2 itself was sufficient to induce actin reorganization and cell scattering in MDCK cells. While Y23E-AnxA2, but not Y23A-AnxA2, enhanced Src- or hepatocyte growth factor (HGF)-induced cell scattering, short hairpin RNA-mediated knockdown of AnxA2 inhibited both v-Src- and HGF-induced cell scattering. Three-dimensional branching morphogenesis was induced in wild-type-AnxA2-expressing cells only in the presence of HGF, while Y23E-AnxA2 induced HGF-independent branching morphogenesis. Knockdown of AnxA2 prevented lumen formation during cystogenesis. The Y23E-AnxA2-induced scattering was associated with dephosphorylation/activation of the actin-severing protein cofilin. Likewise, inactive S3E-cofilin and constitutively active LIM kinase, a direct upstream kinase of cofilin, inhibited Y23E-AnxA2-induced scattering. Together, our studies indicate an essential role for AnxA2 phosphorylation in regulating cofilin-dependent actin cytoskeletal dynamics in the context of cell scattering and branching morphogenesis

A potential role for glycated cross-links in abdominal aortic aneurysm disease

BACKGROUND: Diabetes is a risk factor for atherosclerotic disease but negatively associated with the development and progression of abdominal aortic aneurysm (AAA). Advanced glycation end products (AGEs) are increased in diabetes and renders the vascular matrix more resistant to proteolysis. We assessed the concentration of AGEs in AAA biopsies obtained from diabetic and nondiabetic patients and hypothesized that (nonenzymatic) glycation of AAA tissue protects against proteolytic breakdown of collagen. METHODS: AAA biopsies were collected from 30 diabetic and 30 matched nondiabetic AAA patients at the time of open repair. Aortic control samples from 10 nondiabetic and 16 diabetic patients were collected, and concentrations of the AGE cross-link pentosidine was measured. Furthermore, noncross-linking AGEs (adducts), as well as proteolytic enzymes known to play a role in aneurysm development (matrix metalloproteinase [MMP]-2, MMP-9, cathepsin B and S) were quantified. Ex vivo, nondiabetic AAA biopsies were glycated and measured subsequently for collagen type I release. RESULTS: Pentosidine concentrations in AAA wall biopsies were increased in patients with diabetes compared with nondiabetics 9.4 (5.0-13.5) vs 6.0 (2.5-9.6) pmol/μmol lysine (P = .02). Increased pentosidine concentrations were also observed in nonaneurysmatic aortic wall biopsies from diabetic patients. In diabetic AAA vascular wall tissue, pentosidine concentration was negatively correlated with aortic diameter (r = -0.43; P = .02). Ex vivo glycated AAA biopsies were resistant against MMP-induced collagen type I degradation as compared with controls (7.0 vs 10.4 μg/L; P = .02). No differences were observed for AGEs that are not forming cross-links. CONCLUSIONS: These findings suggest that cross-linking AGEs like pentosidine play a protective role in AAA progression in diabetic patients

A potential role for glycated cross-links in abdominal aortic aneurysm disease

BACKGROUND: Diabetes is a risk factor for atherosclerotic disease but negatively associated with the development and progression of abdominal aortic aneurysm (AAA). Advanced glycation end products (AGEs) are increased in diabetes and renders the vascular matrix more resistant to proteolysis. We assessed the concentration of AGEs in AAA biopsies obtained from diabetic and nondiabetic patients and hypothesized that (nonenzymatic) glycation of AAA tissue protects against proteolytic breakdown of collagen. METHODS: AAA biopsies were collected from 30 diabetic and 30 matched nondiabetic AAA patients at the time of open repair. Aortic control samples from 10 nondiabetic and 16 diabetic patients were collected, and concentrations of the AGE cross-link pentosidine was measured. Furthermore, noncross-linking AGEs (adducts), as well as proteolytic enzymes known to play a role in aneurysm development (matrix metalloproteinase [MMP]-2, MMP-9, cathepsin B and S) were quantified. Ex vivo, nondiabetic AAA biopsies were glycated and measured subsequently for collagen type I release. RESULTS: Pentosidine concentrations in AAA wall biopsies were increased in patients with diabetes compared with nondiabetics 9.4 (5.0-13.5) vs 6.0 (2.5-9.6) pmol/μmol lysine (P = .02). Increased pentosidine concentrations were also observed in nonaneurysmatic aortic wall biopsies from diabetic patients. In diabetic AAA vascular wall tissue, pentosidine concentration was negatively correlated with aortic diameter (r = -0.43; P = .02). Ex vivo glycated AAA biopsies were resistant against MMP-induced collagen type I degradation as compared with controls (7.0 vs 10.4 μg/L; P = .02). No differences were observed for AGEs that are not forming cross-links. CONCLUSIONS: These findings suggest that cross-linking AGEs like pentosidine play a protective role in AAA progression in diabetic patients

Cervical disc width index is a reliable parameter and consistent in young growing Dutch Warmblood horses

Intervertebral disc disease, as well as the associated alteration of the radiographic intervertebral disc space width, has been reported in horses. Disc height index (DHI) has proven to be an accurate and objective parameter in other species but data related to this parameter are lacking in horses. Therefore, the aims of this retrospective longitudinal diagnostic accuracy study were (a) to evaluate the reliability of measurements within and between observers of the equine Disc Width Index (EDWI) as a parameter for radiographic equine cervical intervertebral disc space width, and (b) to evaluate the sequential development of the EDWI over time. For this, EDWI from all intervertebral disc spaces between second cervical (C) to first thoracic (Th) vertebrae were obtained in a group of 39 Dutch Warmblood horses at 1, 5, and 18 months of age, by one European College of Veterinary Diagnostic Imaging (ECVDI) board-certified veterinary radiologist (S.V.) and two veterinary students. Bland-Altmann plots and intraclass Correlation Coefficient revealed a good intra- and interobserver agreement. A linear mixed-effect model did reveal that mean EDWI increases significantly toward the caudal cervical spine, but did not differ significantly for a certain location over time or between sexes. Spearman's rank test did show a significant correlation between the vertebral alignment angle induced by different head-neck positions and a normalized EDWI (rho = 0.33,P < .0001). Student'st-test revealed that the presence of C6-C7 transposition of the transverse processes did not influence EDWI significantly. It was concluded that EDWI represents a reliable parameter for equine cervical radiographic intervertebral disc space width. Practical implementation of EDWI warrants monitoring in a group of adult horses while maintaining a standardized head-neck position

Myocardial Ischemia/Reperfusion Injury Is Mediated by Leukocytic Toll-Like Receptor-2 and Reduced by Systemic Administration of a Novel Anti-Toll-Like Receptor-2 Antibody

BACKGROUND: Reperfusion therapy for myocardial infarction is hampered by detrimental inflammatory responses partly via Toll-like receptor (TLR) activation. Targeting TLR signaling may optimize reperfusion therapy and enhance cell survival and heart function after myocardial infarction. Here, we evaluated the role of TLR2 as a therapeutic target using a novel monoclonal anti-TLR2 antibody. METHOD AND RESULTS: Mice underwent 30 minutes of ischemia followed by reperfusion. Compounds were administered 5 minutes before reperfusion. Cardiac function and dimensions were assessed at baseline and 28 days after infarction with 9.4-T mouse magnetic resonance imaging. Saline and IgG isotype treatment resulted in 34.5 + or - 3.3% and 31.4 + or - 2.7% infarction, respectively. Bone marrow transplantation experiments between wild-type and TLR2-null mice revealed that final infarct size is determined by circulating TLR2 expression. A single intravenous bolus injection of anti-TLR2 antibody reduced infarct size to 18.9 + or - 2.2% (P=0.001). Compared with saline-treated mice, anti-TLR2-treated mice exhibited less expansive remodeling (end-diastolic volume 68.2 + or - 2.5 versus 76.8 + or - 3.5 microL; P=0.046) and preserved systolic performance (ejection fraction 51.0 + or - 2.1% versus 39.9 + or - 2.2%, P=0.009; systolic wall thickening 3.3 + or - 6.0% versus 22.0 + or - 4.4%, P=0.038). Anti-TLR2 treatment significantly reduced neutrophil, macrophage, and T-lymphocyte infiltration. Furthermore, tumor necrosis factor-alpha, interleukin-1alpha, granulocyte macrophage colony-stimulating factor, and interleukin-10 were significantly reduced, as were phosphorylated c-jun N-terminal kinase, phosphorylated p38 mitogen-activated protein kinase, and caspase 3/7 activity levels. CONCLUSIONS: Circulating TLR2 expression mediates myocardial ischemia/reperfusion injury. Antagonizing TLR2 just 5 minutes before reperfusion reduces infarct size and preserves cardiac function and geometry. Anti-TLR2 therapy exerts its action by reducing leukocyte influx, cytokine production, and proapoptotic signaling. Hence, monoclonal anti-TLR2 antibody is a potential candidate as an adjunctive for reperfusion therapy in patients with myocardial infarction.status: publishe

Statins Promote Cardiac Infarct Healing by Modulating Endothelial Barrier Function Revealed by Contrast-Enhanced Magnetic Resonance Imaging

Objective The endothelium has a crucial role in wound healing, acting as a barrier to control transit of leukocytes. Endothelial barrier function is impaired in atherosclerosis preceding myocardial infarction (MI). Besides lowering lipids, statins modulate endothelial function. Here, we noninvasively tested whether statins affect permeability at the inflammatory (day 3) and the reparative (day 7) phase of infarct healing post-MI using contrast-enhanced cardiac magnetic resonance imaging (MRI). Approach and Results Noninvasive permeability mapping by MRI after MI in C57BL/6, atherosclerotic ApoE(-/-), and statin-treated ApoE(-/-) mice was correlated to subsequent left ventricular outcome by structural and functional cardiac MRI. Ex vivo histology, flow cytometry, and quantitative polymerase chain reaction were performed on infarct regions. Increased vascular permeability at ApoE(-/-) infarcts was observed compared with C57BL/6 infarcts, predicting enhanced left ventricular dilation at day 21 post-MI by MRI volumetry. Statin treatment improved vascular barrier function at ApoE(-/-) infarcts, indicated by reduced permeability. The infarcted tissue of ApoE(-/-) mice 3 days post-MI displayed an unbalanced Vegfa(vascular endothelial growth factor A)/Angpt1 (angiopoetin-1) expression ratio (explaining leakage-prone vessels), associated with higher amounts of CD45(+) leukocytes and inflammatory LY6C(hi) monocytes. Statins reversed the unbalanced Vegfa/Angpt1 expression, normalizing endothelial barrier function at the infarct and blocking the augmented recruitment of inflammatory leukocytes in statin-treated ApoE(-/-) mice. Conclusions Statins lowered permeability and reduced the transit of unfavorable inflammatory leukocytes into the infarcted tissue, consequently improving left ventricular outcom