Integrated systems biology approach identifies gene targets for endothelial dysfunction.

Endothelial dysfunction (ED) is critical in the development and progression of cardiovascular (CV) disorders, yet effective therapeutic targets for ED remain elusive due to limited understanding of its underlying molecular mechanisms. To address this gap, we employed a systems biology approach to identify potential targets for ED. Our study combined multi omics data integration, with siRNA screening, high content imaging and network analysis to prioritise key ED genes and identify a pro- and anti-ED network. We found 26 genes that, upon silencing, exacerbated the ED phenotypes tested, and network propagation identified a pro-ED network enriched in functions associated with inflammatory responses. Conversely, 31 genes ameliorated ED phenotypes, pointing to potential ED targets, and the respective anti-ED network was enriched in hypoxia, angiogenesis and cancer-related processes. An independent screen with 17 drugs found general agreement with the trends from our siRNA screen and further highlighted DUSP1, IL6 and CCL2 as potential candidates for targeting ED. Overall, our results demonstrate the potential of integrated system biology approaches in discovering disease-specific candidate drug targets for endothelial dysfunction.

Endothelial protein disulfide isomerase A1 enhances membrane stiffness and platelet-endothelium interaction in hyperglycemia via SLC3A2 and LAMC1.

BACKGROUND: Diabetes carries an increased risk of cardiovascular disease and thromboembolic events. Upon endothelial dysfunction, platelets bind to endothelial cells to precipitate thrombus formation; however, it is unclear which surface proteins regulate platelet-endothelium interaction. We and others have shown that peri/epicellular protein disulfide isomerase A1 (pecPDI) influences the adhesion and migration of vascular cells. OBJECTIVES: We investigated whether pecPDI regulates adhesion-related molecules on the surface of endothelial cells and platelets that influence the binding of these cells in hyperglycemia. METHODS: Immunofluorescence was used to assess platelet-endothelium interaction in vitro, cytoskeleton reorganization, and focal adhesions. Hydrogen peroxide production was assessed via Amplex Red assays (ThermoFisher Scientific). Cell biophysics was assessed using atomic force microscopy. Secreted proteins of interest were identified through proteomics (secretomics), and targets were knocked down using small interfering RNA. Protein disulfide isomerase A1 (PDI) contribution was assessed using whole-cell PDI or pecPDI inhibitors or small interfering RNA. RESULTS: Platelets of healthy donors adhered more onto hyperglycemic human umbilical vein endothelial cells (HUVECs). Endothelial, but not platelet, pecPDI regulated this effect. Hyperglycemic HUVECs showed marked cytoskeleton reorganization, increased H2O2 production, and elongated focal adhesions. Indeed, hyperglycemic HUVECs were stiffer compared with normoglycemic cells. PDI and pecPDI inhibition reversed the abovementioned processes in hyperglycemic cells. A secretomics analysis revealed 8 proteins secreted in a PDI-dependent manner by hyperglycemic cells. Among these, we showed that genetic deletion of LAMC1 and SLC3A2 decreased platelet-endothelium interaction and did not potentiate the effects of PDI inhibitors. CONCLUSION: Endothelial pecPDI regulates platelet-endothelium interaction in hyperglycemia through adhesion-related proteins and alterations in endothelial membrane biophysics.

Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch.

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the reduced long-term patency compared to arterial conduits. The abrupt increase of hemodynamic stress associated with the graft arterialization results in vascular damage, especially the endothelium, that may influence the low patency of the saphenous vein graft (SVG). Here, we describe the isolation, characterization, and expansion of human saphenous vein endothelial cells (hSVECs). Cells isolated by collagenase digestion display the typical cobblestone morphology and express endothelial cell markers CD31 and VE-cadherin. To assess the mechanical stress influence, protocols were used in this study to investigate the two main physical stimuli, shear stress and stretch, on arterialized SVGs. hSVECs are cultured in a parallel plate flow chamber to produce shear stress, showing alignment in the direction of the flow and increased expression of KLF2, KLF4, and NOS3. hSVECs can also be cultured in a silicon membrane that allows controlled cellular stretch mimicking venous (low) and arterial (high) stretch. Endothelial cells' F-actin pattern and nitric oxide (NO) secretion are modulated accordingly by the arterial stretch. In summary, we present a detailed method to isolate hSVECs to study the influence of hemodynamic mechanical stress on an endothelial phenotype.

Cysteine and glycine-rich protein 3 (Crp3) as a critical regulator of elastolysis, inflammation, and smooth muscle cell apoptosis in abdominal aortic aneurysm development.

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease for which surgical or endovascular repair are the only currently available therapeutic strategies. The development of AAA involves the breakdown of elastic fibers (elastolysis), infiltration of inflammatory cells, and apoptosis of smooth muscle cells (SMCs). However, the specific regulators governing these responses remain unknown. We previously demonstrated that Cysteine and glycine-rich protein 3 (Crp3) sensitizes SMCs to apoptosis induced by stretching. Building upon this finding, we aimed to investigate the influence of Crp3 on elastolysis and apoptosis during AAA development. Using the elastase-CaCl2 rat model, we observed an increase in Crp3 expression, aortic diameter, and a reduction in wall thickness in wild type rats. In contrast, Crp3-/- rats exhibited a decreased incidence of AAA, with minimal or no changes in aortic diameter and thickness. Histopathological analysis revealed the absence of SMC apoptosis and degradation of elastic fibers in Crp3-/- rats, accompanied by reduced inflammation and diminished proteolytic capacity in Crp3-/- SMCs and bone marrow-derived macrophages. Collectively, our findings provide evidence that Crp3 plays a crucial role in AAA development by modulating elastolysis, inflammation, and SMC apoptosis. These results underscore the potential significance of Crp3 in the context of AAA progression and offer new insights into therapeutic targets for this disease.

A modified hydrogel production protocol to decrease cellular content.

PURPOSE: To analyze the cytotoxicity and cell in porcine-derived decellularized skin matrix. METHODS: We analyzed the effect of multiple decellularization processes by histological analysis, DNA quantification, and flow cytometry. Subsequently, we analyzed the most appropriate hydrogel concentration to minimize cytotoxicity on fibroblast culture and to maximize cell proliferation. RESULTS: After the fourth decellularization, the DNA quantification showed the lowest DNA concentration (< 50 ng/mg). Histological analysis showed no cell components in the hydrogel. Moreover, hematoxylin and eosin showed a heterogeneous structure of collagen fibers. The best hydrogel concentration ranged from 3 to 25%, and there was no significant difference between the 24 hours and seven days. CONCLUSIONS: The process of hydrogel production was effective for removing cells and DNA elements. The best hydrogel concentration ranged from 3 to 25%.

Aerobic training improves NAFLD markers and insulin resistance through AMPK-PPAR-α signaling in obese mice.

Liver steatosis is one of the main drivers for the development of whole-body insulin resistance. Conversely, aerobic training (AT) has been suggested as non-pharmacological tool to improve liver steatosis, however, the underlying molecular mechanism remains unclear. Therefore, the aim of this study was to analyze the effect of 8-weeks AT in non-alcoholic liver disease (NAFLD) outcomes in obese mice. Male C57BL/6 J wild type (WT) were fed with standard (SD) or high-fat diet (HFD) for 12-weeks. Another group fed with HFD underwent 8-weeks of AT (60% of maximum velocity), initiated at the 5th week of experimental protocol. We measured metabolic, body composition parameters, protein and gene expression inflammatory and metabolic mediators. We found that AT attenuates the weight gain, but not body fat accumulation. AT improved triacylglycerol and non-esterified fatty acid plasma concentrations, and also whole-body insulin resistance. Regarding NAFLD, AT decreased the progression of macrovesicular steatosis and inflammation through the upregulation of AMPK Thr172 phosphorylation and PPAR-α protein expression. Moreover, although no effects of intervention in PPAR-γ protein concentration were observed, we found increased levels of its target genes Cd36 and Scd1 in exercised group, demonstrating augmented transcriptional activity. AT reduced liver cytokines concentrations, such as TNF-α, IL-10, MCP-1 and IL-6, regardless of increased Ser536 NF-κB phosphorylation. In fact, none of the interventions regulated NF-κB target genes Il1b and Cccl2, demonstrating its low transcriptional activity. Therefore, we conclude that AT attenuates the progression of liver macrovesicular steatosis and inflammation through AMPK-PPAR-α signaling and PPAR-γ activation, respectively, improving insulin resistance in obese mice.

Environmental control of mammary carcinoma cell expansion by acidification and spheroid formation in vitro.

Breast cancer is the leading cause of cancer death among women worldwide. Like other cancers, mammary carcinoma progression involves acidification of the tumor microenvironment, which is an important factor for cancer detection and treatment strategies. However, the effects of acidity on mammary carcinoma cell morphology and phenotype have not been thoroughly characterized. Here, we evaluated fundamental effects of environmental acidification on mammary carcinoma cells in standard two-dimensional cultures and three-dimensional spheroids. Acidification decreased overall mammary carcinoma cell viability, while increasing their resistance to the anthracycline doxorubicin. Environmental acidification also increased extracellular vesicle production by mammary carcinoma cells. Conditioned media containing these vesicles appeared to increase fibroblast motility. Acidification also increased mammary carcinoma cell motility when cultured with fibroblasts in spheroids. Taken together, results from this study suggest that environmental acidification induces drug resistance and extracellular vesicle production by mammary carcinoma cells that promote tumor expansion.