Endothelial Cell Insulin Signaling Regulates CXCR4 (C-X-C Motif Chemokine Receptor 4) and Limits Leukocyte Adhesion to Endothelium.

BACKGROUND: An activated, proinflammatory endothelium is a key feature in the development of complications of obesity and type 2 diabetes and can be caused by insulin resistance in endothelial cells. METHODS: We analyzed primary human endothelial cells by RNA sequencing to discover novel insulin-regulated genes and used endothelial cell culture and animal models to characterize signaling through CXCR4 (C-X-C motif chemokine receptor 4) in endothelial cells. RESULTS: CXCR4 was one of the genes most potently regulated by insulin, and this was mediated by PI3K (phosphatidylinositol 3-kinase), likely through FoxO1, which bound to the CXCR4 promoter. CXCR4 mRNA in CD31+ cells was 77% higher in mice with diet-induced obesity compared with lean controls and 37% higher in db/db mice than db/+ controls, consistent with upregulation of CXCR4 in endothelial cell insulin resistance. SDF-1 (stromal cell-derived factor-1)-the ligand for CXCR4-increased leukocyte adhesion to cultured endothelial cells. This effect was lost after deletion of CXCR4 by gene editing while 80% of the increase was prevented by treatment of endothelial cells with insulin. In vivo microscopy of mesenteric venules showed an increase in leukocyte rolling after intravenous injection of SDF-1, but most of this response was prevented in transgenic mice with endothelial overexpression of IRS-1 (insulin receptor substrate-1). CONCLUSIONS: Endothelial cell insulin signaling limits leukocyte/endothelial cell interaction induced by SDF-1 through downregulation of CXCR4. Improving insulin signaling in endothelial cells or inhibiting endothelial CXCR4 may reduce immune cell recruitment to the vascular wall or tissue parenchyma in insulin resistance and thereby help prevent several vascular complications.

The transcriptional coregulator CITED2 suppresses expression of IRS-2 and impairs insulin signaling in endothelial cells.

Endothelial cell insulin resistance contributes to the development of vascular complications in diabetes. Hypoxia-inducible factors (HIFs) modulate insulin sensitivity, and we have previously shown that a negative regulator of HIF activity, CREB-binding protein/p300 (CBP/p300) interacting transactivator-2 (CITED2), is increased in the vasculature of people with type 2 diabetes. Therefore, we examined whether CITED2 regulates endothelial insulin sensitivity. In endothelial cells isolated from mice with a "floxed" mutation in the Cited2 gene, loss of CITED2 markedly enhanced insulin-stimulated Akt phosphorylation without altering extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation. Similarly, insulin-stimulated Akt phosphorylation was increased in aortas of mice with endothelial-specific deletion of CITED2. Consistent with these observations, loss of CITED2 in endothelial cells increased insulin-stimulated endothelial nitric oxide synthase phosphorylation, Vegfa expression, and cell proliferation. Endothelial cells lacking CITED2 exhibited an increase in insulin receptor substrate (IRS)-2 protein, a key mediator of the insulin signaling cascade, whereas IRS-1 was unchanged. Conversely, overexpression of CITED2 in endothelial cells decreased IRS-2 protein by 55% without altering IRS-1, resulting in impaired insulin-stimulated Akt phosphorylation and Vegfa expression. Overexpression of HIF-2α significantly increased activity of the Irs2 promoter, and coexpression of CITED2 abolished this increase. Moreover, chromatin immunoprecipitation (ChIP) showed that loss of CITED2 increased occupancy of p300, a key component of the HIF transcriptional complex, on the Irs2 promoter. Together, these results show that CITED2 selectively inhibits endothelial insulin signaling and action through the phosphoinositide 3-kinase (PI3K)/Akt pathway via repression of HIF-dependent IRS-2 expression. CITED2 is thus a promising target to improve endothelial insulin sensitivity and prevent the vascular complications of diabetes.NEW & NOTEWORTHY Endothelial cell insulin resistance is a major contributor to the development of diabetic complications. In this study, we have shown that CITED2, a transcriptional coregulator, inhibits endothelial insulin signaling through the PI3K/Akt pathway via repression of HIF-dependent IRS-2 expression, and that deletion of CITED2 enhances insulin signaling. Thus, CITED2 represents a novel and promising target to improve insulin sensitivity in endothelial cells and prevent vascular complications in diabetes.

γδ T cell dichotomy with opposing cytotoxic and wound healing functions in human solid tumors.

γδ T cells are important tissue-resident, innate T cells that are critical for tissue homeostasis. γδ cells are associated with positive prognosis in most tumors; however, little is known about their heterogeneity in human cancers. Here, we phenotyped innate and adaptive cells in human colorectal (CRC) and endometrial cancer. We found striking differences in γδ subsets and function in tumors compared to normal tissue, and in the γδ subsets present in tumor types. In CRC, an amphiregulin (AREG)-producing subset emerges, while endometrial cancer is infiltrated by cytotoxic cells. In humanized CRC models, tumors induced this AREG phenotype in Vδ1 cells after adoptive transfer. To exploit the beneficial roles of γδ cells for cell therapy, we developed an expansion method that enhanced cytotoxic function and boosted metabolic flexibility, while eliminating AREG production, achieving greater tumor infiltration and tumor clearance. This method has broad applications in cellular therapy as an 'off-the-shelf' treatment option.

Dysregulation of TSP2-Rac1-WAVE2 axis in diabetic cells leads to cytoskeletal disorganization, increased cell stiffness, and dysfunction.

Fibroblasts are a major cell population that perform critical functions in the wound healing process. In response to injury, they proliferate and migrate into the wound space, engaging in extracellular matrix (ECM) production, remodeling, and contraction. However, there is limited knowledge of how fibroblast functions are altered in diabetes. To address this gap, several state-of-the-art microscopy techniques were employed to investigate morphology, migration, ECM production, 2D traction, 3D contraction, and cell stiffness. Analysis of cell-derived matrix (CDM) revealed that diabetic fibroblasts produce thickened and less porous ECM that hindered migration of normal fibroblasts. In addition, diabetic fibroblasts were found to lose spindle-like shape, migrate slower, generate less traction force, exert limited 3D contractility, and have increased cell stiffness. These changes were due, in part, to a decreased level of active Rac1 and a lack of co-localization between F-actin and Waskott-Aldrich syndrome protein family verprolin homologous protein 2 (WAVE2). Interestingly, deletion of thrombospondin-2 (TSP2) in diabetic fibroblasts rescued these phenotypes and restored normal levels of active Rac1 and WAVE2-F-actin co-localization. These results provide a comprehensive view of the extent of diabetic fibroblast dysfunction, highlighting the regulatory role of the TSP2-Rac1-WAVE2-actin axis, and describing a new function of TSP2 in regulating cytoskeleton organization.

Longitudinal analysis of invariant natural killer T cell activation reveals a cMAF-associated transcriptional state of NKT10 cells.

Innate T cells, including CD1d-restricted invariant natural killer T (iNKT) cells, are characterized by their rapid activation in response to non-peptide antigens, such as lipids. While the transcriptional profiles of naive, effector, and memory adaptive T cells have been well studied, less is known about the transcriptional regulation of different iNKT cell activation states. Here, using single-cell RNA-sequencing, we performed longitudinal profiling of activated murine iNKT cells, generating a transcriptomic atlas of iNKT cell activation states. We found that transcriptional signatures of activation are highly conserved among heterogeneous iNKT cell populations, including NKT1, NKT2, and NKT17 subsets, and human iNKT cells. Strikingly, we found that regulatory iNKT cells, such as adipose iNKT cells, undergo blunted activation and display constitutive enrichment of memory-like cMAF+ and KLRG1+ populations. Moreover, we identify a conserved cMAF-associated transcriptional network among NKT10 cells, providing novel insights into the biology of regulatory and antigen-experienced iNKT cells.

Elevated Thrombospondin 2 Contributes to Delayed Wound Healing in Diabetes.

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 current 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 patients with diabetes. 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 with diabetic control mice, they exhibited significantly improved healing, characterized by accelerated reepithelialization 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 nuclear factor-κB signaling to elevate TSP2 expression. Our studies demonstrate that hyperglycemia-induced TSP2 expression contributes to impaired healing in diabetes.

Decellularized materials derived from TSP2-KO mice promote enhanced neovascularization and integration in diabetic wounds.

Decellularized biologic scaffolds are gaining popularity over synthetic biomaterials as naturally derived materials capable of promoting improved healing. Nevertheless, the most widely used biologic material - acellular dermal matrix (ADM) - exhibits slow repopulation and remodeling, which prevents integration. Additionally, engineering control of these materials is limited because they require a natural source for their production. In the current report, we demonstrate the feasibility of using genetically engineered animals to create decellularized biologic scaffolds with favorable extracellular matrix (ECM) properties. Specifically, we utilized skin from thrombospondin (TSP)-2 KO mice to derive various decellularized products. Scanning electron microscopy and mechanical testing showed that TSP-2 KO ADM exhibited an altered structure and a reduction in elastic modulus and ultimate tensile strength, respectively. When a powdered form of KO ADM was implanted subcutaneously, it was able to promote enhanced vascularization over WT. Additionally, when implanted subcutaneously, intact slabs of KO ADM were populated by higher number of host cells when compared to WT. In vitro studies confirmed the promigratory properties of KO ADM. Specifically, degradation products released by pepsin digestion of KO ADM induced greater cell migration than WT. Moreover, cell-derived ECM from TSP-2 null fibroblasts was more permissive to fibroblast migration. Finally, ADMs were implanted in a diabetic wound model to examine their ability to accelerate wound healing. KO ADM exhibited enhanced remodeling and vascular maturation, indicative of efficient integration. Overall, we demonstrate that genetic manipulation enables engineered ECM-based materials with increased regenerative potential.

Redox Signaling in Diabetic Wound Healing Regulates Extracellular Matrix Deposition.

SIGNIFICANCE: Impaired wound healing is a major complication of diabetes, and can lead to development of chronic foot ulcers in a significant number of patients. Despite the danger posed by poor healing, very few specific therapies exist, leaving patients at risk of hospitalization, amputation, and further decline in overall health. Recent Advances: Redox signaling is a key regulator of wound healing, especially through its influence on the extracellular matrix (ECM). Normal redox signaling is disrupted in diabetes leading to several pathological mechanisms that alter the balance between reactive oxygen species (ROS) generation and scavenging. Importantly, pathological oxidative stress can alter ECM structure and function. CRITICAL ISSUES: There is limited understanding of the specific role of altered redox signaling in the diabetic wound, although there is evidence that ROS are involved in the underlying pathology. FUTURE DIRECTIONS: Preclinical studies of antioxidant-based therapies for diabetic wound healing have yielded promising results. Redox-based therapeutics constitute a novel approach for the treatment of wounds in diabetes patients that deserve further investigation. Antioxid. Redox Signal. 27, 823-838.

Production of decellularized porcine lung scaffolds for use in tissue engineering.

There is a growing body of work dedicated to producing acellular lung scaffolds for use in regenerative medicine by decellularizing donor lungs of various species. These scaffolds typically undergo substantial matrix damage due to the harsh conditions required to remove cellular material (e.g., high pH, strong detergents), lengthy processing times, or pre-existing tissue contamination from microbial colonization. In this work, a new decellularization technique is described that maintains the global tissue architecture, key matrix components, mechanical composition and cell-seeding potential of lung tissue while effectively removing resident cellular material. Acellular lung scaffolds were produced from native porcine lungs using a combination of Triton X-100 and sodium deoxycholate (SDC) at low concentrations in 24 hours. We assessed the effect of matrix decellularization by measuring residual DNA, biochemical composition, mechanical characteristics, tissue architecture, and recellularization capacity.