Pik3c3 Expression Profiling in the Mouse Kidney and Its Role in Proximal Tubule Cell Physiology.

The class 3 phosphatidylinositol 3-kinase (Pik3c3) plays critical roles in regulating autophagy, endocytosis, and nutrient sensing, but its expression profile in the kidney remains undefined. Recently, we validated a Pik3c3 antibody through immunofluorescence staining of kidney tissues from cell type-specific Pik3c3 knockout mice. Immunohistochemistry unveiled significant disparities in Pik3c3 expression levels across various kidney cell types. Notably, renal interstitial cells exhibit minimal Pik3c3 expression. Further, co-immunofluorescence staining, utilizing nephron segment- or cell type-specific markers, revealed nearly undetectable levels of Pik3c3 expression in glomerular mesangial cells and endothelial cells. Intriguingly, although podocytes exhibit the highest Pik3c3 expression levels among all kidney cell types, the renal proximal tubule cells (RPTCs) express the highest level of Pik3c3 among all renal tubules. RPTCs are known to express the highest level of the epidermal growth factor receptor (EGFR) in adult kidneys; however, the role of Pik3c3 in EGFR signaling within RPTCs remains unexplored. Therefore, we conducted additional cell culture studies. The results demonstrated that Pik3c3 inhibition significantly delayed EGF-stimulated EGFR degradation and the termination of EGFR signaling in RPTCs. Mechanistically, Pik3c3 inhibition surprisingly did not affect the initial endocytosis process but instead impeded the lysosomal degradation of EGFR. In summary, this study defines, for the first time, the expression profile of Pik3c3 in the mouse kidney and also highlights a pivotal role of Pik3c3 in the proximal tubule cells. These findings shed light on the intricate mechanisms underlying Pik3c3-mediated regulation of EGFR signaling, providing valuable insights into the role of Pik3c3 in renal cell physiology.

Mechanisms of pulmonary endothelial barrier dysfunction in acute lung injury and acute respiratory distress syndrome.

Endothelial cells (ECs) form a semi-permeable barrier between the interior space of blood vessels and the underlying tissues. Pulmonary endothelial barrier integrity is maintained through coordinated cellular processes involving receptors, signaling molecules, junctional complexes, and protein-regulated cytoskeletal reorganization. In acute lung injury (ALI) or its more severe form acute respiratory distress syndrome (ARDS), the loss of endothelial barrier integrity secondary to endothelial dysfunction caused by severe pulmonary inflammation and/or infection leads to pulmonary edema and hypoxemia. Pro-inflammatory agonists such as histamine, thrombin, bradykinin, interleukin 1ß, tumor necrosis factor α, vascular endothelial growth factor, angiopoietin-2, and platelet-activating factor, as well as bacterial toxins and reactive oxygen species, cause dynamic changes in cytoskeletal structure, adherens junction disorganization, and detachment of vascular endothelial cadherin (VE-cadherin) from the actin cytoskeleton, leading to an increase in endothelial permeability. Endothelial interactions with leukocytes, platelets, and coagulation enhance the inflammatory response. Moreover, inflammatory infiltration and the associated generation of pro-inflammatory cytokines during infection cause EC death, resulting in further compromise of the structural integrity of lung endothelial barrier. Despite the use of potent antibiotics and aggressive intensive care support, the mortality of ALI is still high, because the mechanisms of pulmonary EC barrier disruption are not fully understood. In this review, we summarized recent advances in the studies of endothelial cytoskeletal reorganization, inter-endothelial junctions, endothelial inflammation, EC death, and endothelial repair in ALI and ARDS, intending to shed some light on the potential diagnostic and therapeutic targets in the clinical management of the disease.

Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy.

BACKGROUND: Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. Apart from Parkin, little is known about additional Ub (ubiquitin) ligases that mediate mitochondrial ubiquitination and turnover, particularly in highly metabolically active organs such as the heart. METHODS: In this study, we have combined in silico analysis and biochemical assay to identify CRL (cullin-RING ligase) 5 as a mitochondrial Ub ligase. We generated cardiomyocytes and mice lacking RBX2 (RING-box protein 2; also known as SAG [sensitive to apoptosis gene]), a catalytic subunit of CRL5, to understand the effects of RBX2 depletion on mitochondrial ubiquitination, mitophagy, and cardiac function. We also performed proteomics analysis and RNA-sequencing analysis to define the impact of loss of RBX2 on the proteome and transcriptome. RESULTS: RBX2 and CUL (cullin) 5, 2 core components of CRL5, localize to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, increased cardiomyocyte cell death, and has a global impact on the mitochondrial proteome. In vivo, deletion of the Rbx2 gene in adult mouse hearts suppressed mitophagic activity, provoked accumulation of damaged mitochondria in the myocardium, and disrupted myocardial metabolism, leading to the rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. The action of RBX2 in mitochondria is not dependent on Parkin, and Parkin gene deletion had no impact on the onset and progression of cardiomyopathy in RBX2-deficient hearts. Furthermore, RBX2 controls the stability of PINK1 (PTEN-induced kinase 1) in mitochondria. CONCLUSIONS: These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that regulates mitophagy and cardiac homeostasis in a Parkin-independent, PINK1-dependent manner.

Transgenic Overexpression of HDAC9 Promotes Adipocyte Hypertrophy, Insulin Resistance and Hepatic Steatosis in Aging Mice.

Histone deacetylase (HDAC) 9 is a negative regulator of adipogenic differentiation, which is required for maintenance of healthy adipose tissues. We reported that HDAC9 expression is upregulated in adipose tissues during obesity, in conjunction with impaired adipogenic differentiation, adipocyte hypertrophy, insulin resistance, and hepatic steatosis, all of which were alleviated by global genetic deletion of Hdac9. Here, we developed a novel transgenic (TG) mouse model to test whether overexpression of Hdac9 is sufficient to induce adipocyte hypertrophy, insulin resistance, and hepatic steatosis in the absence of obesity. HDAC9 TG mice gained less body weight than wild-type (WT) mice when fed a standard laboratory diet for up to 40 weeks, which was attributed to reduced fat mass (primarily inguinal adipose tissue). There was no difference in insulin sensitivity or glucose tolerance in 18-week-old WT and HDAC9 TG mice; however, at 40 weeks of age, HDAC9 TG mice exhibited impaired insulin sensitivity and glucose intolerance. Tissue histology demonstrated adipocyte hypertrophy, along with reduced numbers of mature adipocytes and stromovascular cells, in the HDAC9 TG mouse adipose tissue. Moreover, increased lipids were detected in the livers of aging HDAC9 TG mice, as evaluated by oil red O staining. In conclusion, the experimental aging HDAC9 TG mice developed adipocyte hypertrophy, insulin resistance, and hepatic steatosis, independent of obesity. This novel mouse model may be useful in the investigation of the impact of Hdac9 overexpression associated with metabolic and aging-related diseases.

PDZ-Binding Kinase, a Novel Regulator of Vascular Remodeling in Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is high blood pressure in the lungs that originates from structural changes in small resistance arteries. A defining feature of PAH is the inappropriate remodeling of pulmonary arteries (PA) leading to right ventricle failure and death. Although treatment of PAH has improved, the long-term prognosis for patients remains poor, and more effective targets are needed. METHODS: Gene expression was analyzed by microarray, RNA sequencing, quantitative polymerase chain reaction, Western blotting, and immunostaining of lung and isolated PA in multiple mouse and rat models of pulmonary hypertension (PH) and human PAH. PH was assessed by digital ultrasound, hemodynamic measurements, and morphometry. RESULTS: Microarray analysis of the transcriptome of hypertensive rat PA identified a novel candidate, PBK (PDZ-binding kinase), that was upregulated in multiple models and species including humans. PBK is a serine/threonine kinase with important roles in cell proliferation that is minimally expressed in normal tissues but significantly increased in highly proliferative tissues. PBK was robustly upregulated in the medial layer of PA, where it overlaps with markers of smooth muscle cells. Gain-of-function approaches show that active forms of PBK increase PA smooth muscle cell proliferation, whereas silencing PBK, dominant negative PBK, and pharmacological inhibitors of PBK all reduce proliferation. Pharmacological inhibitors of PBK were effective in PH reversal strategies in both mouse and rat models, providing translational significance. In a complementary genetic approach, PBK was knocked out in rats using CRISPR/Cas9 editing, and loss of PBK prevented the development of PH. We found that PBK bound to PRC1 (protein regulator of cytokinesis 1) in PA smooth muscle cells and that multiple genes involved in cytokinesis were upregulated in experimental models of PH and human PAH. Active PBK increased PRC1 phosphorylation and supported cytokinesis in PA smooth muscle cells, whereas silencing or dominant negative PBK reduced cytokinesis and the number of cells in the G2/M phase of the cell cycle. CONCLUSIONS: PBK is a newly described target for PAH that is upregulated in proliferating PA smooth muscle cells, where it contributes to proliferation through changes in cytokinesis and cell cycle dynamics to promote medial thickening, fibrosis, increased PA resistance, elevated right ventricular systolic pressure, right ventricular remodeling, and PH.

The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy.

Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. While the role of ubiquitin (Ub) ligase PARKIN in mitophagy has been extensively studied, increasing evidence suggests the existence of PARKIN-independent mitophagy in highly metabolically active organs such as the heart. Here, we identify a crucial role for Cullin-RING Ub ligase 5 (CRL5) in basal mitochondrial turnover in cardiomyocytes. CRL5 is a multi-subunit Ub ligase comprised by the catalytic RING box protein RBX2 (also known as SAG), scaffold protein Cullin 5 (CUL5), and a substrate-recognizing receptor. Analysis of the mitochondrial outer membrane-interacting proteome uncovered a robust association of CRLs with mitochondria. Subcellular fractionation, immunostaining, and immunogold electron microscopy established that RBX2 and Cul5, two core components of CRL5, localizes to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, and increased cell death in cardiomyocytes. In vivo , deletion of the Rbx2 gene in adult mouse hearts suppressed mitophagic activity, provoked accumulation of damaged mitochondria in the myocardium, and disrupted myocardial metabolism, leading to rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. Notably, the action of RBX2 in mitochondria is not dependent on PARKIN, and PARKIN gene deletion had no impact on the onset and progression of cardiomyopathy in RBX2-deficient hearts. Furthermore, RBX2 controls the stability of PINK1 in mitochondria. Proteomics and biochemical analyses further revealed a global impact of RBX2 deficiency on the mitochondrial proteome and identified several mitochondrial proteins as its putative substrates. These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that controls mitophagy under physiological conditions in a PARKIN-independent, PINK1-dependent manner, thereby regulating cardiac homeostasis.

Zinc-Dependent Histone Deacetylases in Lung Endothelial Pathobiology.

A monolayer of endothelial cells (ECs) lines the lumen of blood vessels and, as such, provides a semi-selective barrier between the blood and the interstitial space. Compromise of the lung EC barrier due to inflammatory or toxic events may result in pulmonary edema, which is a cardinal feature of acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS). The EC functions are controlled, at least in part, via epigenetic mechanisms mediated by histone deacetylases (HDACs). Zinc-dependent HDACs represent the largest group of HDACs and are activated by Zn2+. Members of this HDAC group are involved in epigenetic regulation primarily by modifying the structure of chromatin upon removal of acetyl groups from histones. In addition, they can deacetylate many non-histone histone proteins, including those located in extranuclear compartments. Recently, the therapeutic potential of inhibiting zinc-dependent HDACs for EC barrier preservation has gained momentum. However, the role of specific HDAC subtypes in EC barrier regulation remains largely unknown. This review aims to provide an update on the role of zinc-dependent HDACs in endothelial dysfunction and its related diseases. We will broadly focus on biological contributions, signaling pathways and transcriptional roles of HDACs in endothelial pathobiology associated mainly with lung diseases, and we will discuss the potential of their inhibitors for lung injury prevention.

NADPH oxidase 1 promotes hepatic steatosis in obese mice and is abrogated by augmented skeletal muscle mass.

Exercise as a lifestyle modification is a frontline therapy for nonalcoholic fatty liver disease (NAFLD), but how components of exercise attenuate steatosis is unclear. To uncouple the effect of increased muscle mass from weight loss in obesity, myostatin knockout mice were bred on a lean and obese db/db background. Myostatin deletion increases gastrocnemius (Gastrocn.) mass and reduces hepatic steatosis and hepatic sterol regulatory element binding protein 1 (Srebp1) expression in obese mice, with no impact on adiposity or body weight. Interestingly, hypermuscularity reduces hepatic NADPH oxidase 1 (Nox1) expression but not NADPH oxidase 4 (Nox4) in db/db mice. To evaluate a deterministic function of Nox1 on steatosis, Nox1 knockout mice were bred on a lean and db/db background. NOX1 deletion significantly attenuates hepatic oxidant stress, steatosis, and Srebp1 programming in obese mice to parallel hypermuscularity, with no improvement in adiposity, glucose control, or hypertriglyceridemia to suggest off-target effects. Directly assessing the role of NOX1 on SREBP1, insulin (Ins)-mediated SREBP1 expression was significantly increased in either NOX1, NADPH oxidase organizer 1 (NOXO1), and NADPH oxidase activator 1 (NOXA1) or NOX5-transfected HepG2 cells versus ?-galactosidase control virus, indicating superoxide is the key mechanistic agent for the actions of NOX1 on SREBP1. Metabolic Nox1 regulators were evaluated using physiological, genetic, and diet-induced animal models that modulated upstream glucose and insulin signaling, identifying hyperinsulinemia as the key metabolic derangement explaining Nox1-induced steatosis in obesity. GEO data revealed that hepatic NOX1 predicts steatosis in obese humans with biopsy-proven NAFLD. Taken together, these data suggest that hypermuscularity attenuates Srebp1 expression in db/db mice through a NOX1-dependent mechanism.NEW & NOTEWORTHY This study documents a novel mechanism by which changes in body composition, notably increased muscle mass, protect against fatty liver disease. This mechanism involves NADPH oxidase 1 (NOX1), an enzyme that increases superoxide and increases insulin signaling, leading to increased fat accumulation in the liver. NOX1 may represent a new early target for preventing fatty liver to stave off later liver diseases such as cirrhosis or liver cancer.

Impact of housing temperature on adipose tissue HDAC9 expression and adipogenic differentiation in high fat-fed mice.

OBJECTIVE: Impaired adipogenic differentiation exacerbates metabolic disease in obesity. This study reported that high-fat diet (HFD)-fed mice housed at thermoneutrality exhibited impaired adipogenic differentiation, attributed to increased expression of histone deacetylase 9 (HDAC9). However, the impact of HFD on adipogenic differentiation is reportedly variable, possibly reflecting divergent environmental conditions such as housing temperature. METHODS: C57BL/6J (wild-type [WT]) mice were housed at either thermoneutral (28-30°C) or ambient (20-22°C) temperature and fed HFD or chow diet (CD) for 12 weeks. For acute exposure experiments, WT or transient receptor potential cation channel subfamily M member 8 (TRPM8) knockout mice housed under thermoneutrality were acutely exposed to ambient temperature for 6 to 24 h. RESULTS: WT mice fed HFD and housed at thermoneutrality, compared with ambient temperature, gained more weight despite reduced food intake. They likewise exhibited increased inguinal adipose tissue HDAC9 expression and reduced adipogenic differentiation in vitro and in vivo compared with CD-fed mice. Conversely, HFD-fed mice housed at ambient temperature exhibited minimal change in adipose HDAC9 expression or adipogenic differentiation. Acute exposure of WT mice to ambient temperature reduced adipose HDAC9 expression independent of sympathetic ß-adrenergic signaling via a TRPM8-dependent mechanism. CONCLUSIONS: Adipose HDAC9 expression is temperature sensitive, regulating adipogenic differentiation in HFD-fed mice housed under thermoneutrality.

Integrative multiomics analysis of neointima proliferation in human saphenous vein: implications for bypass graft disease.

Introduction: Human saphenous veins (SV) are widely used as grafts in coronary artery bypass (CABG) surgery but often fail due to neointima proliferation (NP). NP involves complex interplay between vascular smooth muscle cells (VSMC) and fibroblasts. Little is known, however, regarding the transcriptomic and proteomic dynamics of NP. Here, we performed multi-omics analysis in an ex vivo tissue culture model of NP in human SV procured for CABG surgery. Methods and results: Histological examination demonstrated significant elastin degradation and NP (indicated by increased neointima area and neointima/media ratio) in SV subjected to tissue culture. Analysis of data from 73 patients suggest that the process of SV adaptation and NP may differ according to sex and body mass index. RNA sequencing confirmed upregulation of pro-inflammatory and proliferation-related genes during NP and identified novel processes, including increased cellular stress and DNA damage responses, which may reflect tissue trauma associated with SV harvesting. Proteomic analysis identified upregulated extracellular matrix-related and coagulation/thrombosis proteins and downregulated metabolic proteins. Spatial transcriptomics detected transdifferentiating VSMC in the intima on the day of harvesting and highlighted dynamic alterations in fibroblast and VSMC phenotype and behavior during NP. Specifically, we identified new cell subpopulations contributing to NP, including SPP1 + , LGALS3 + VSMC and MMP2 + , MMP14 + fibroblasts. Conclusion: Dynamic alterations of gene and protein expression occur during NP in human SV. Identification of the human-specific molecular and cellular mechanisms may provide novel insight into SV bypass graft disease.

Calbindin 2-specific deletion of arginase 2 preserves visual function after optic nerve crush.

We previously found that global deletion of the mitochondrial enzyme arginase 2 (A2) limits optic nerve crush (ONC)-induced neuronal death. Herein, we examined the cell-specific role of A2 in this pathology by studies using wild type (WT), neuronal-specific calbindin 2 A2 KO (Calb2cre/+ A2 f/f), myeloid-specific A2 KO (LysMcre/+ A2f/f), endothelial-specific A2 KO (Cdh5cre/+ A2f/f), and floxed controls. We also examined the impact of A2 overexpression on mitochondrial function in retinal neuronal R28 cells. Immunolabeling showed increased A2 expression in ganglion cell layer (GCL) neurons of WT mice within 6 h-post injury and inner retinal neurons after 7 days. Calb2 A2 KO mice showed improved neuronal survival, decreased TUNEL-positive neurons, and improved retinal function compared to floxed littermates. Neuronal loss was unchanged by A2 deletion in myeloid or endothelial cells. We also found increased expression of neurotrophins (BDNF, FGF2) and improved survival signaling (pAKT, pERK1/2) in Calb2 A2 KO retinas within 24-hour post-ONC along with suppression of inflammatory mediators (IL1ß, TNFα, IL6, and iNOS) and apoptotic markers (cleavage of caspase3 and PARP). ONC increased GFAP and Iba1 immunostaining in floxed controls, and Calb2 A2 KO dampened this effect. Overexpression of A2 in R28 cells increased Drp1 expression, and decreased mitochondrial respiration, whereas ABH-induced inhibition of A2 decreased Drp1 expression and improved mitochondrial respiration. Finally, A2 overexpression or excitotoxic treatment with glutamate significantly impaired mitochondrial function in R28 cells as shown by significant reductions in basal respiration, maximal respiration, and ATP production. Further, glutamate treatment of A2 overexpressing cells did not induce further deterioration in their mitochondrial function, indicating that A2 overexpression or glutamate insult induce comparable alterations in mitochondrial function. Our data indicate that neuronal A2 expression is neurotoxic after injury, and A2 deletion in Calb2 expressing neurons limits ONC-induced retinal neurodegeneration and improves visual function.

Type 1 Diabetes Impairs Endothelium-Dependent Relaxation Via Increasing Endothelial Cell Glycolysis Through Advanced Glycation End Products, PFKFB3, and Nox1-Mediated Mechanisms.

BACKGROUND: Type 1 diabetes (T1D) is a major cause of endothelial dysfunction. Although cellular bioenergetics has been identified as a new regulator of vascular function, whether glycolysis, the primary bioenergetic pathway in endothelial cells (EC), regulates vascular tone and contributes to impaired endothelium-dependent relaxation (EDR) in T1D remains unknown. METHODS: Experiments were conducted in Akita mice with intact or selective deficiency in EC PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3), the main regulator of glycolysis. Seahorse analyzer and myography were employed to measure glycolysis and mitochondrial respiration, and EDR, respectively, in aortic explants. EC PFKFB3 (Ad-PFKFB3) and glycolysis (Ad-GlycoHi) were increased in situ via adenoviral transduction. RESULTS: T1D increased EC glycolysis and elevated EC expression of PFKFB3 and NADPH oxidase Nox1 (NADPH oxidase homolog 1). Functionally, pharmacological and genetic inhibition of PFKFB3 restored EDR in T1D, while in situ aorta EC transduction with Ad-PFKFB3 or Ad-GlycoHi reproduced the impaired EDR associated with T1D. Nox1 inhibition restored EDR in aortic rings from Akita mice, as well as in Ad-PFKFB3-transduced aorta EC and lactate-treated wild-type aortas. T1D increased the expression of the advanced glycation end product precursor methylglyoxal in the aortas. Exposure of the aortas to methylglyoxal impaired EDR, which was prevented by PFKFB3 inhibition. T1D and exposure to methylglyoxal increased EC expression of HIF1α (hypoxia-inducible factor 1α), whose inhibition blunted methylglyoxal-mediated EC PFKFB3 upregulation. CONCLUSIONS: EC bioenergetics, namely glycolysis, is a new regulator of vasomotion and excess glycolysis, a novel mechanism of endothelial dysfunction in T1D. We introduce excess methylglyoxal, HIF1α, and PFKFB3 as major effectors in T1D-mediated increased EC glycolysis.

A Reappraisal of the Utility of L-012 to Measure Superoxide from Biologically Relevant Sources.

The detection of superoxide anion (O2●-) in biological tissues remains challenging. Barriers to convenient and reproducible measurements include expensive equipment, custom probes, and the need for high sensitivity and specificity. The luminol derivative, L-012, has been used to measure O2●- since 1993 with mixed results and concerns over specificity. The goal of this study was to better define the conditions for use and their specificity. We found that L-012 coupled with depolymerized orthovanadate, a relatively impermeable tyrosine phosphatase inhibitor, yielded a highly sensitive approach to detect extracellular O2●-. In O2●- producing HEK-NOX5 cells, orthovanadate increased L-012 luminescence 100-fold. The combination of L-012 and orthovanadate was highly sensitive, stable, scalable, completely reversed by superoxide dismutase, and selective for O2●- generating NOXes versus NOX4, which produces H2O2. Moreover, there was no signal from cells transfected with NOS3 (NO●) and NOS2(ONOO-). To exclude the effects of altered tyrosine phosphorylation, O2●- was detected using non-enzymatic synthesis with phenazine methosulfate and via novel coupling of L-012 with niobium oxalate, which was less active in inducing tyrosine phosphorylation. Overall, our data shows that L-012 coupled with orthovanadate or other periodic group 5 salts yields a reliable, sensitive, and specific approach to measuring extracellular O2●- in biological systems.

Calpain Promotes LPS-induced Lung Endothelial Barrier Dysfunction via Cleavage of Talin.

Acute lung injury (ALI) is characterized by lung vascular endothelial cell (EC) barrier compromise resulting in increased endothelial permeability and pulmonary edema. The infection of gram-negative bacteria that produce toxins like LPS is one of the major causes of ALI. LPS activates Toll-like receptor 4, leading to cytoskeleton reorganization, resulting in lung endothelial barrier disruption and pulmonary edema in ALI. However, the signaling pathways that lead to the cytoskeleton reorganization and lung microvascular EC barrier disruption remain largely unexplored. Here we show that LPS induces calpain activation and talin cleavage into head and rod domains and that inhibition of calpain attenuates talin cleavage, RhoA activation, and pulmonary EC barrier disruption in LPS-treated human lung microvascular ECs in vitro and lung EC barrier disruption and pulmonary edema induced by LPS in ALI in vivo. Moreover, overexpression of calpain causes talin cleavage and RhoA activation, myosin light chain (MLC) phosphorylation, and increases in actin stress fiber formation. Furthermore, knockdown of talin attenuates LPS-induced RhoA activation and MLC phosphorylation and increased stress fiber formation and mitigates LPS-induced lung microvascular endothelial barrier disruption. Additionally, overexpression of talin head and rod domains increases RhoA activation, MLC phosphorylation, and stress fiber formation and enhances lung endothelial barrier disruption. Finally, overexpression of cleavage-resistant talin mutant reduces LPS-induced increases in MLC phosphorylation in human lung microvascular ECs and attenuates LPS-induced lung microvascular endothelial barrier disruption. These results provide the first evidence that calpain mediates LPS-induced lung microvascular endothelial barrier disruption in ALI via cleavage of talin.

Galectin-3 Mediates Vascular Dysfunction in Obesity by Regulating NADPH Oxidase 1.

BACKGROUND: Obesity is associated with increased risk of cardiovascular disease, but underlying mechanisms remain elusive. Metabolic dysfunction, especially hyperglycemia, is thought to be a major contributor, but how glucose impacts vascular function is unclear. GAL3 (galectin-3) is a sugar-binding lectin upregulated by hyperglycemia, but its role as a causative mechanism of cardiovascular disease remains poorly understood. Therefore, the objective of this study was to determine the role of GAL3 in regulating microvascular endothelial vasodilation in obesity. METHODS: GAL3 was measured and found to be markedly increased in the plasma of overweight and obese patients, as well as in the microvascular endothelium of diabetic patients. To investigate causative mechanisms in cardiovascular disease, mice deficient in GAL3 were bred with obese db/db mice to generate lean, lean GAL3 knockout, obese, and obese GAL3 knockout genotypes. Endothelial cell-specific GAL3 knockout mice with novel AAV-induced obesity recapitulated whole-body knockout studies to confirm cell specificity. RESULTS: Deletion of GAL3 did not alter body mass, adiposity, or plasma indices of glycemia and lipidemia, but levels of plasma reactive oxygen species as assessed by plasma thiobarbituric acid reactive substances were normalized in obese GAL3 knockout mice. Obese mice exhibited profound endothelial dysfunction and hypertension, both of which were rescued by GAL3 deletion. Isolated microvascular endothelial cells from obese mice had increased expression of NOX1 (nicotinamide adenine dinucleotide phosphate oxidase 1), which we have previously shown to contribute to increased oxidative stress and endothelial dysfunction, which was normalized in microvascular endothelium from mice lacking GAL3. Cell-specific deletion confirmed that endothelial GAL3 regulates obesity-induced NOX1 overexpression and subsequent microvascular function. Furthermore, improvement of metabolic syndrome by increasing muscle mass, improving insulin signaling, or treating with metformin decreased microvascular GAL3, and thereby NOX1, expression levels. CONCLUSIONS: Deletion of GAL3 normalizes microvascular endothelial function in obese db/db mice, likely through a NOX1-mediated mechanism. Pathological levels of GAL3, and in turn NOX1, are amenable to improvements in metabolic status, presenting a potential therapeutic target to ameliorate pathological cardiovascular consequences of obesity.

Hepatocyte-specific disruption of soluble epoxide hydrolase attenuates abdominal aortic aneurysm formation: novel role of the liver in aneurysm pathogenesis.

Introduction: Inflammation is a key pathogenic feature of abdominal aortic aneurysm (AAA). Soluble epoxide hydrolase (sEH) is a pro-inflammatory enzyme that converts cytochrome P450-derived epoxides of fatty acids to the corresponding diols, and pharmacological inhibition of sEH prevented AAA formation. Both cytochrome P450 enzymes and sEH are highly expressed in the liver. Here, we investigated the role of hepatic sEH in AAA using a selective pharmacological inhibitor of sEH and hepatocyte-specific Ephx2 (which encodes sEH gene) knockout (KO) mice in two models of AAA [angiotensin II (AngII) infusion and calcium chloride (CaCl 2 ) application]. Methods and results: sEH expression and activity were strikingly higher in mouse liver compared with aorta and further increased the context of AAA, in conjunction with elevated expression of the transcription factor Sp1 and the epigenetic regulator Jarid1b, which have been reported to positively regulate sEH expression. Pharmacological sEH inhibition, or liver-specific sEH disruption, achieved by crossing sEH floxed mice with albumin-cre mice, prevented AAA formation in both models, concomitant with reduced expression of hepatic sEH as well as complement factor 3 (C3) and serum amyloid A (SAA), liver-derived factors linked to AAA formation. Moreover, sEH antagonism markedly reduced C3 and SAA protein accumulation in the aortic wall. Co-incubation of liver ex vivo with aneurysm-prone aorta resulted in induction of sEH in the liver, concomitant with upregulation of Sp1, Jarid1b, C3 and SAA gene expression, suggesting that the aneurysm-prone aorta secretes factors that activate sEH and downstream inflammatory signaling in the liver. Using an unbiased proteomic approach, we identified a number of dysregulated proteins [ e.g., plastin-2, galectin-3 (gal-3), cathepsin S] released by aneurysm-prone aorta as potential candidate mediators of hepatic sEH induction. Conclusion: We provide the first direct evidence of the liver's role in orchestrating AAA via the enzyme sEH. These findings not only provide novel insight into AAA pathogenesis, but they have potentially important implications with regard to developing effective medical therapies for AAA.

Serine/Threonine Protein Phosphatases 1 and 2A in Lung Endothelial Barrier Regulation.

Vascular barrier dysfunction is characterized by increased permeability and inflammation of endothelial cells (ECs), which are prominent features of acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and sepsis, and a major complication of the SARS-CoV-2 infection and COVID-19. Functional impairment of the EC barrier and accompanying inflammation arises due to microbial toxins and from white blood cells of the lung as part of a defensive action against pathogens, ischemia-reperfusion or blood product transfusions, and aspiration syndromes-based injury. A loss of barrier function results in the excessive movement of fluid and macromolecules from the vasculature into the interstitium and alveolae resulting in pulmonary edema and collapse of the architecture and function of the lungs, and eventually culminates in respiratory failure. Therefore, EC barrier integrity, which is heavily dependent on cytoskeletal elements (mainly actin filaments, microtubules (MTs), cell-matrix focal adhesions, and intercellular junctions) to maintain cellular contacts, is a critical requirement for the preservation of lung function. EC cytoskeletal remodeling is regulated, at least in part, by Ser/Thr phosphorylation/dephosphorylation of key cytoskeletal proteins. While a large body of literature describes the role of phosphorylation of cytoskeletal proteins on Ser/Thr residues in the context of EC barrier regulation, the role of Ser/Thr dephosphorylation catalyzed by Ser/Thr protein phosphatases (PPases) in EC barrier regulation is less documented. Ser/Thr PPases have been proposed to act as a counter-regulatory mechanism that preserves the EC barrier and opposes EC contraction. Despite the importance of PPases, our knowledge of the catalytic and regulatory subunits involved, as well as their cellular targets, is limited and under-appreciated. Therefore, the goal of this review is to discuss the role of Ser/Thr PPases in the regulation of lung EC cytoskeleton and permeability with special emphasis on the role of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) as major mammalian Ser/Thr PPases. Importantly, we integrate the role of PPases with the structural dynamics of the cytoskeleton and signaling cascades that regulate endothelial cell permeability and inflammation.

Galectin-3 Mediates Vascular Dysfunction in Obesity by Regulating NADPH Oxidase 1.

Rationale: Obesity increases the risk of cardiovascular disease (CVD) through mechanisms that remain incompletely defined. Metabolic dysfunction, especially hyperglycemia, is thought to be a major contributor but how glucose impacts vascular function is unclear. Galectin-3 (GAL3) is a sugar binding lectin upregulated by hyperglycemia but its role as a causative mechanism of CVD remains poorly understood. Objective: To determine the role of GAL3 in regulating microvascular endothelial vasodilation in obesity. Methods and Results: GAL3 was markedly increased in the plasma of overweight and obese patients, as well as in the microvascular endothelium of diabetic patients. To investigate a role for GAL3 in CVD, mice deficient in GAL3 were bred with obese db/db mice to generate lean, lean GAL3 knockout (KO), obese, and obese GAL3 KO genotypes. GAL3 KO did not alter body mass, adiposity, glycemia or lipidemia, but normalized elevated markers of reactive oxygen species (TBARS) in plasma. Obese mice exhibited profound endothelial dysfunction and hypertension, both of which were rescued by GAL3 deletion. Isolated microvascular endothelial cells (EC) from obese mice had increased NOX1 expression, which we have previously shown to contribute to increased oxidative stress and endothelial dysfunction, and NOX1 levels were normalized in EC from obese mice lacking GAL3. EC-specific GAL3 knockout mice made obese using a novel AAV-approach recapitulated whole-body knockout studies, confirming that endothelial GAL3 drives obesity-induced NOX1 overexpression and endothelial dysfunction. Improved metabolism through increased muscle mass, enhanced insulin signaling, or metformin treatment, decreased microvascular GAL3 and NOX1. GAL3 increased NOX1 promoter activity and this was dependent on GAL3 oligomerization. Conclusions: Deletion of GAL3 normalizes microvascular endothelial function in obese db/db mice, likely through a NOX1-mediated mechanism. Pathological levels of GAL3 and in turn, NOX1, are amenable to improvements in metabolic status, presenting a potential therapeutic target to ameliorate pathological cardiovascular consequences of obesity.