Loss of integrity of the epithelial/mucosal barrier in the small intestine has been associated with different pathologies that originate and/or develop in the gastrointestinal tract. We showed recently that mucin, the main protein in the mucus layer, is disrupted during early periods of intestinal ischemia. This event is accompanied by entry of pancreatic digestive enzymes into the intestinal wall. We hypothesize that the mucin-containing mucus layer is the main barrier preventing digestive enzymes from contacting the epithelium. Mucin breakdown may render the epithelium accessible to pancreatic enzymes, causing its disruption and increased permeability. The objective of this study was to investigate the role of mucin as a protection for epithelial integrity and function. A rat model of 30 min splanchnic arterial occlusion (SAO) was used to study the degradation of two mucin isoforms (mucin 2 and 13) and two epithelial membrane proteins (E-cadherin and toll-like receptor 4, TLR4). In addition, the role of digestive enzymes in mucin breakdown was assessed in this model by luminal inhibition with acarbose, tranexamic acid, or nafamostat mesilate. Furthermore, the protective effect of the mucin layer against trypsin-mediated disruption of the intestinal epithelium was studied in vitro. Rats after SAO showed degradation of mucin 2 and fragmentation of mucin 13, which was not prevented by protease inhibition. Mucin breakdown was accompanied by increased intestinal permeability to FITC-dextran as well as degradation of E-cadherin and TLR4. Addition of mucin to intestinal epithelial cells in vitro protected against trypsin-mediated degradation of E-cadherin and TLR4 and reduced permeability of FITC-dextran across the monolayer. These results indicate that mucin plays an important role in the preservation of the mucosal barrier and that ischemia but not digestive enzymes disturbs mucin integrity, while digestive enzymes actively mediate epithelial cell disruption.

Chronic venous disease (CVD) has a range of clinical presentations, including tortuous, distended veins in lower extremities, increasing skin pigmentation, and in severe cases ulceration of the affected skin. Venous insufficiency, a precursor to CVD characterized by improper return of blood from the lower extremities to the heart, must be studied in its earliest stages at a time when preventative measures could be applied in man. This underscores the need for basic research into biomarkers and genetic predisposing factors affecting the progression of venous disease. Investigation over the past decade has yielded insight into these specific genetic, cellular and molecular mechanisms underlying the development of venous disease. Among the many advances include the elucidation of an increasing role for matrix metalloproteinases as important mediators of the degenerative process involved with venous insufficiency. This may be preceded by an inflammatory process which further contributes to venular degeneration and endothelial dysfunction seen in advanced presentation of disease. Furthermore, genomic analyses have shed light upon temporal expression patterns of matrix remodeling proteins in diseased tissue samples. In this review we examine some of the current findings surrounding cellular, molecular and genetic advances in delineating the etiology of chronic venous disease.

Atherogenesis involves an interplay of inflammation, tissue remodeling and cellular transdifferentiation (CTD), making it especially difficult to precisely delineate its pathophysiology. Here we use single-cell RNA sequencing and systems-biology approaches to analyze the transcriptional profiles of vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) in calcified atherosclerotic core (AC) plaques and patient-matched proximal adjacent (PA) portions of carotid artery tissue from patients undergoing carotid endarterectomy. Our results reveal an anatomic distinction whereby PA cells express inflammatory mediators, while cells expressing matrix-secreting genes occupy a majority of the AC region. Systems biology analysis indicates that inflammation in PA ECs and VSMCs may be driven by TNFa signaling. Furthermore, we identify POSTN, SPP1 and IBSP in AC VSMCs, and ITLN1, SCX and S100A4 in AC ECs as possible candidate drivers of CTD in the atherosclerotic core. These results establish an anatomic framework for atherogenesis which forms the basis for exploration of a site-specific strategy for disruption of disease progression.

Background: Matrix metalloproteinases (MMP) and VEGFR2 often coexist in many settings, but their interactions are unknown. Results: MMP-1 stimulates VEGFR2 up-regulation in endothelial cells. Conclusion: MMP-1-stimulated cells have elevated intracellular signaling and proliferate at a faster rate than unstimulated cells. Significance: A novel mechanism is uncovered whereby MMP-1 is able to sensitize endothelial cell functions.

Atherogenesis involves an interplay of inflammation, tissue remodeling and cellular transdifferentiation (CTD), making it especially difficult to precisely delineate its pathophysiology. Here we examine the single-cell transcriptome of entire atherosclerotic core (AC) plaques and patient-matched proximal adjacent (PA) portions of carotid artery tissue from patients undergoing carotid endarterectomy. We use a novel tissue dissociation strategy, single-cell RNA sequencing, and systems-biology approaches to analyze the transcriptional profiles of six main cell populations and identify key gene drivers of pathogenic biological processes in vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). Our results reveal an anatomic continuum whereby PA cells promote and respond to inflammatory processes and eventually transition through CTD into matrix-secreting cells in the AC. Inflammatory signaling in PA ECs is driven by IL6, while TNFa signaling defines inflammation in both PA ECs and VSMCs. Furthermore, we identify POSTN, SPP1 and IBSP in AC VSMCs, and ITLN1, SCX and S100A4 in AC ECs as key drivers of CTD in the atherosclerotic core. These results establish an anatomic framework for atherogenesis and suggest a site-specific strategy for disruption of disease progression.

Obesity and related morbidities pose a major health threat. Obesity is associated with increased blood concentrations of the anorexigenic hormone leptin; however, obese individuals are resistant to its anorexigenic effects. We examined the phenomenon of reduced leptin signaling in a high-fat diet-induced obesity model in mice. Obesity promoted matrix metalloproteinase-2 (Mmp-2) activation in the hypothalamus, which cleaved the leptin receptor's extracellular domain and impaired leptin-mediated signaling. Deletion of Mmp-2 restored leptin receptor expression and reduced circulating leptin concentrations in obese mice. Lentiviral delivery of short hairpin RNA to silence in the hypothalamus of wild-type mice prevented leptin receptor cleavage and reduced fat accumulation. In contrast, lentiviral delivery of in the hypothalamus of mice promoted leptin receptor cleavage and higher body weight. In a genetic mouse model of obesity, transduction of cleavage-resistant leptin receptor in the hypothalamus reduced the rate of weight gain compared to uninfected mice or mice infected with the wild-type receptor. Immunofluorescence analysis showed that astrocytes and agouti-related peptide neurons were responsible for Mmp-2 secretion in mice fed a high-fat diet. These results suggest a mechanism for leptin resistance through activation of Mmp-2 and subsequent cleavage of the extracellular domain of the leptin receptor.

In bowel ischemia, impaired mucosal integrity may allow intestinal pancreatic enzyme products to become systemic and precipitate irreversible shock and death. This can be attenuated by pancreatic enzyme inhibition in the small bowel lumen. It is unresolved, however, whether ischemically-mediated mucosal disruption is the key event allowing pancreatic enzyme products systemic access, and whether intestinal digestive enzyme activity in concert with increased mucosal permeability leads to shock in the absence of ischemia. To test this possibility, the small intestinal lumen of non-ischemic rats was perfused for two hours with either digestive enzymes, a mucin disruption strategy (i.e., mucolytics) designed to increase mucosal permeability, or both, and animals were observed for shock. Digestive enzymes perfused included trypsin, chymotrypsin, elastase, amylase and lipase. Control (n=6) and experimental animals perfused with pancreatic enzymes only (n=6) or single enzymes (n=3 for each of the five enzyme groups) maintained stable hemodynamics. After mucin disruption using a combination of enteral N-acetylcysteine, atropine, and increased flow rates, rats (n=6) developed mild hypotension (p<0.001 compared to groups perfused with pancreatic enzymes only after 90 minutes) and increased intestinal permeability to intralumenally perfused FITC-dextrans-20kD (p<0.05) compared to control and enzyme-only groups, but there were no deaths. All animals perfused with both digestive enzymes and subjected to mucin disruption (n=6) developed hypotension and increased intestinal permeability (p<0.001 after 90 minutes). Pancreatic enzymes were measured in the intestinal wall of both groups subjected to mucin disruption, but not in the enzyme-only or control groups. Depletion of plasma protease inhibitors was found only in animals perfused with pancreatic enzymes plus mucin disruption, implicating increased permeability and intralumenal pancreatic enzyme egress in this group. These experiments demonstrate that increased bowel permeability via mucin disruption in the presence of pancreatic enzymes can induce shock and increase systemic protease activation in the absence of ischemia, implicating bowel mucin disruption as a key event in early ischemia. Digestive enzymes and their products, if allowed to penetrate the gut wall may trigger multiorgan failure and death.

Aims De-differentiation and activation of pro-inflammatory pathways are key transitions vascular smooth muscle cells (SMCs) make during atherogenesis. Here, we explored the upstream regulators of this ‘atherogenic transition’. Methods and results Genome-wide sequencing studies, including ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) and RNA-seq, were performed on cells isolated from both murine SMC-lineage tracing models of atherosclerosis and human atherosclerotic lesions. At the bulk level, alterations in chromatin accessibility were associated with the atherogenic transitioning of lesional SMCs, especially in relation to genes that govern differentiation status and complement-dependent inflammation. Using computational biology, we observed that a transcription factor previously related to coronary artery disease, ATF3 (Activating transcription factor 3), was predicted to be an upstream regulator of genes altered during the transition. At the single-cell level, our results indicated that ATF3 is a key repressor of SMC transitioning towards the subset of cells that promote vascular inflammation by activating the complement cascade. The expression of ATF3 and complement component C3 were negatively correlated in SMCs from human atherosclerotic lesions, suggesting translational relevance. Phenome-wide association studies indicated that genetic variation that results in reduced expression of ATF3 is correlated with an increased risk for atherosclerosis, and the expression of ATF3 was significantly downregulated in humans with advanced vascular disease. Conclusion Our study indicates that the plasticity of atherosclerotic SMCs may in part be explained by dynamic changes in their chromatin architecture, which in turn may contribute to their maladaptive response to inflammation-induced stress. Translational perspective The recent CANTOS and COLCOT trials have shown that targeting inflammatory pathways lowers the risk of major adverse cardiovascular events. However, more specific targets are needed to avoid immunosuppressive side effects. Our data identify an upstream regulator of pro-inflammatory SMCs, ATF3, which is involved in the initial atherogenic transitioning of lesional SMCs. Restoring ATF3 activity may prevent the de-differentiation of SMCs and offer a novel translational approach for the suppression of complement-dependent inflammation in atherosclerotic lesions.