Faculty Bibliography

BACKGROUND AND AIMS/OBJECTIVE:COVID-19 is associated with liver injury and elevated IL-6. We hypothesized that IL-6 trans-signaling in liver sinusoidal endothelial cells (LSECs) leads to endotheliopathy (a proinflammatory and procoagulant state) and liver injury in COVID-19. METHODS:Coagulopathy, endotheliopathy, and ALT were retrospectively analyzed in a subset (n=68), followed by a larger cohort(n=3,780) of COVID-19 patients. Liver histology from 43 COVID-19 patients was analyzed for endotheliopathy and its relationship to liver injury. Primary human LSECs were used to establish the IL-6 trans-signaling mechanism. RESULTS:Factor VIII, fibrinogen, D-dimer, vWF activity/antigen (biomarkers of coagulopathy/endotheliopathy) were significantly elevated in COVID-19 patients with liver injury (elevated ALT). IL-6 positively correlated with vWF antigen(P=0.02), factor VIII activity(P=0.02), and D-dimer(P<0.0001). On liver histology, COVID-19 patients with elevated ALT had significantly increased vWF and platelet staining, supporting a link between liver injury, coagulopathy, and endotheliopathy. Intralobular neutrophils positively correlated with platelet(P<0.0001) and vWF(P<0.01) staining, and IL-6 levels positively correlated with vWF staining(P<0.01). IL-6 trans-signaling leads to increased expression of procoagulant (Factor VIII, vWF) and proinflammatory factors, increased cell surface vWF(P<0.01), and increased platelet attachment in LSECs. These effects were blocked by soluble gp130 (IL-6 trans-signaling inhibitor), JAK inhibitor Ruxolitinib, and STAT1/3 siRNA knockdown. Hepatocyte fibrinogen expression was increased by the supernatant of LSECs subjected to IL-6 trans-signaling. CONCLUSION/CONCLUSIONS:COVID-19 is associated with coagulopathy and endotheliopathy in the liver endothelium driven by IL-6 trans-signaling, a possible mechanism of liver injury. LAY SUMMARY/UNASSIGNED:Patients with SARS-CoV-2 infection often have liver injury, but why this occurs remains unknown. High levels of interleukin-6 (IL-6) and its circulating receptor, which form a complex to induce inflammatory signals, have been observed in COVID-19 patients. This paper demonstrates that the IL-6 signaling complex causes harmful changes to liver sinusoidal endothelial cells and may promote blood clotting and contribute to liver injury.

Background Proteinuria and glomerular segmental fibrosis are inevitable complications of diabetic nephropathy though their mechanisms are poorly understood. Understanding the clinical characteristics and pathogenesis of proteinuria and glomerular segmental fibrosis in diabetic nephropathy is, therefore, urgently needed for patient management of this severe disease. Methods and Results Diabetes mellitus was induced in podocyte-specific glucocorticoid receptor knockout (GR^PKO) mice and control littermates by administration of streptozotocin. Primary podocytes were isolated and subjected to analysis of Wnt signaling and fatty acid metabolism. Conditioned media from primary podocytes was transferred to glomerular endothelial cells. Histologic analysis of kidneys from diabetic GR^PKO mice showed worsened fibrosis, increased collagen deposition, and glomerulomegaly indicating severe glomerular fibrosis. Higher expression of transforming growth factor-βR1 and β-catenin and suppressed expression of carnitine palmitoyltransferase 1A in nephrin-positive cells were found in the kidneys of diabetic GR^PKO mice. Podocytes isolated from diabetic GR^PKO mice demonstrated significantly higher profibrotic gene expression and suppressed fatty acid oxidation compared with controls. Administration of a Wnt inhibitor significantly improved the fibrotic features in GR^PKO mice. The glomerular endothelium of diabetic GR^PKO mice demonstrated the features of endothelial-to-mesenchymal transition. Moreover, endothelial cells treated with conditioned media from podocytes lacking GR showed increased expression of α-smooth muscle actin, transforming growth factor-βR1 and β-catenin levels. Conclusions These data demonstrate that loss of podocyte GR leads to upregulation of Wnt signaling and disruption in fatty acid metabolism. Podocyte-endothelial cell crosstalk, mediated through GR, is important for glomerular homeostasis, and its disruption likely contributes to diabetic nephropathy.

Hepatic uptake and biosynthesis of fatty acids (FA), as well as the partitioning of FA into oxidative, storage, and secretory pathways are tightly regulated processes. Dysregulation of one or more of these processes can promote excess hepatic lipid accumulation, ultimately leading to systemic metabolic dysfunction. Angiopoietin-like-4 (ANGPTL4) is a secretory protein that inhibits lipoprotein lipase (LPL) and modulates triacylglycerol (TAG) homeostasis. To understand the role of ANGPTL4 in liver lipid metabolism under normal and high-fat fed conditions, we generated hepatocyte specific Angptl4 mutant mice (Hmut). Using metabolic turnover studies, we demonstrate that hepatic Angptl4 deficiency facilitates catabolism of TAG-rich lipoprotein (TRL) remnants in the liver via increased hepatic lipase (HL) activity, which results in a significant reduction in circulating TAG and cholesterol levels. Consequently, depletion of hepatocyte Angptl4 protects against diet-induce obesity, glucose intolerance, liver steatosis, and atherogenesis. Mechanistically, we demonstrate that loss of Angptl4 in hepatocytes promotes FA uptake which results in increased FA oxidation, ROS production, and AMPK activation. Finally, we demonstrate the utility of a targeted pharmacologic therapy that specifically inhibits Angptl4 gene expression in the liver and protects against diet-induced obesity, dyslipidemia, glucose intolerance, and liver damage, which likely occurs via increased HL activity. Notably, this novel inhibition strategy does not cause any of the deleterious effects previously observed with neutralizing antibodies.

Endothelial cells play a key role in the regulation of disease. Defective regulation of endothelial cell homeostasis may cause mesenchymal activation of other endothelial cells or neighboring cell types, and in both cases contributes to organ fibrosis. Regulatory control of endothelial cell homeostasis is not well studied. Diabetes accelerates renal fibrosis in mice lacking the endothelial glucocorticoid receptor (GR), compared to control mice. Hypercholesterolemia further enhances severe renal fibrosis. The fibrogenic phenotype in the kidneys of diabetic mice lacking endothelial GR is associated with aberrant cytokine and chemokine reprogramming, augmented Wnt signaling and suppression of fatty acid oxidation. Both neutralization of IL-6 and Wnt inhibition improve kidney fibrosis by mitigating mesenchymal transition. Conditioned media from endothelial cells from diabetic mice lacking endothelial GR stimulate Wnt signaling-dependent epithelial-to-mesenchymal transition in tubular epithelial cells from diabetic controls. These data demonstrate that endothelial GR is an essential antifibrotic molecule in diabetes.

Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence. Previously, using an unbiased screen, we identified ALK-1 as a high-capacity receptor for low-density lipoprotein (LDL) in endothelial cells that mediates its transcytosis in a nondegradative manner. Here we examine the crosstalk between BMP-9 and LDL and how it influences their interactions with ALK-1. Treatment of endothelial cells with BMP-9 triggers the extensive endocytosis of ALK-1, and it is mediated by caveolin-1 (CAV-1) and dynamin-2 (DNM2) but not clathrin heavy chain. Knockdown of CAV-1 reduces BMP-9-mediated internalization of ALK-1, BMP-9-dependent signaling and gene expression. Similarly, treatment of endothelial cells with LDL reduces BMP-9-induced SMAD1/5 phosphorylation and gene expression and silencing of CAV-1 and DNM2 diminishes LDL-mediated ALK-1 internalization. Interestingly, BMP-9-mediated ALK-1 internalization strongly re-duces LDL transcytosis to levels seen with ALK-1 deficiency. Thus, BMP-9 levels can control cell surface levels of ALK-1, via CAV-1, to regulate both BMP-9 signaling and LDL transcytosis.

The thymus is a primary lymphoid organ necessary for optimal T cell development. Here, we show that liver X receptors (LXRs)-a class of nuclear receptors and transcription factors with diverse functions in metabolism and immunity-critically contribute to thymic integrity and function. LXRαβ-deficient mice develop a fatty, rapidly involuting thymus and acquire a shrunken and prematurely immunoinhibitory peripheral T cell repertoire. LXRαβ's functions are cell specific, and the resulting phenotypes are mutually independent. Although thymic macrophages require LXRαβ for cholesterol efflux, thymic epithelial cells (TECs) use LXRαβ for self-renewal and thymocytes for negative selection. Consequently, TEC-derived LXRαβ protects against homeostatic premature involution and orchestrates thymic regeneration following stress, while thymocyte-derived LXRαβ limits cell disposal during negative selection and confers heightened sensitivity to experimental autoimmune encephalomyelitis. These results identify three distinct but complementary mechanisms by which LXRαβ governs T lymphocyte education and illuminate LXRαβ's indispensable roles in adaptive immunity.

Obesity is a top public health concern, and a molecule that safely treats obesity is urgently needed. Disulfiram (known commercially as Antabuse), an FDA-approved treatment for chronic alcohol addiction, exhibits anti-inflammatory properties and helps protect against certain types of cancer. Here, we show that in mice disulfiram treatment prevented body weight gain and abrogated the adverse impact of an obesogenic diet on insulin responsiveness while mitigating liver steatosis and pancreatic islet hypertrophy. Additionally, disulfiram treatment reversed established diet-induced obesity and metabolic dysfunctions in middle-aged mice. Reductions in feeding efficiency and increases in energy expenditure were associated with body weight regulation in response to long-term disulfiram treatment. Loss of fat tissue and an increase in liver fenestrations were also observed in rats on disulfiram. Given the potent anti-obesogenic effects in rodents, repurposing disulfiram in the clinic could represent a new strategy to treat obesity and its metabolic comorbidities.

The etiology of aortic aneurysms is poorly understood, but it is associated with atherosclerosis, hypercholesterolemia, and abnormal transforming growth factor β (TGF-β) signaling in smooth muscle. Here, we investigated the interactions between these different factors in aortic aneurysm development and identified a key role for smooth muscle cell (SMC) reprogramming into a mesenchymal stem cell (MSC)-like state. SMC-specific ablation of TGF-β signaling in Apoe^-/- mice on a hypercholesterolemic diet led to development of aortic aneurysms exhibiting all the features of human disease, which was associated with transdifferentiation of a subset of contractile SMCs into an MSC-like intermediate state that generated osteoblasts, chondrocytes, adipocytes, and macrophages. This combination of medial SMC loss with marked increases in non-SMC aortic cell mass induced exuberant growth and dilation of the aorta, calcification and ossification of the aortic wall, and inflammation, resulting in aneurysm development.

There is an unmet need and urgency to find safe and effective anti-obesity interventions. Our recent study in mice fed on obesogenic diet found that treatment with the alcohol aversive drug disulfiram reduced feeding efficiency and led to a decrease in body weight and an increase in energy expenditure. The intervention with disulfiram improved glucose tolerance and insulin sensitivity, and mitigated metabolic dysfunctions in various organs through poorly defined mechanisms. Here, integrated analysis of transcriptomic and proteomic data from mouse and rat livers unveiled comparable signatures in response to disulfiram, revealing pathways associated with lipid and energy metabolism, redox, and detoxification. In cell culture, disulfiram was found to be a potent activator of autophagy, the malfunctioning of which has negative consequences on metabolic regulation. Thus, repurposing disulfiram may represent a potent strategy to combat obesity.

Impaired wound healing is a major complication of diabetes and despite the associated risks, treatment strategies for diabetic wounds remain limited. This is due, in part, to an incomplete understanding of the underlying pathological mechanisms, including the effects of hyperglycemia on components of the extracellular matrix (ECM). In the present study, we explored whether the expression of thrombospondin-2 (TSP2), a matricellular protein with a demonstrated role in response to injury, was associated with delayed healing in diabetes. First, we found that TSP2 expression was elevated in diabetic mice and skin from diabetic patients. Then, to determine the contribution of TSP2 to impaired healing in diabetes, we developed a novel diabetic TSP2-deficient model. Though the TSP2 deficient mice developed obesity and hyperglycemia comparable to diabetic control mice, they exhibited significantly improved healing, characterized by accelerated re-epithelialization and increased granulation tissue formation, fibroblast migration, and blood vessel maturation. We further found that hyperglycemia increased TSP2 expression in fibroblasts, the major cellular source of TSP2 in wounds. Mechanistically, high glucose increased activation of the hexosamine pathway and NF-κB signaling to elevate TSP2 expression. Our studies demonstrate that hyperglycemia-induced TSP2 expression contributes to impaired healing in diabetes.