Robert Karoly Batori

Obesity and type 2 diabetes (T2D) increase cardiovascular risk, largely due to altered metabolic state. An early consequence of T2D/obesity is the loss of endothelial function and impaired nitric oxide (NO) signaling. In blood vessels, endothelial nitric oxide synthase (eNOS) synthesizes NO to maintain vessel homeostasis. The biological actions of NO are compromised by superoxide that is generated by NADPH oxidases (NOXs). Herein we investigated how altered metabolism affects superoxide/NO balance in obesity. We found that eNOS expression and NO bioavailability are significantly decreased in endothelial cells (ECs) from T2D patients and animal models of obesity. In parallel, PFKFB3, a key glycolytic regulatory enzyme, is significantly increased in ECs of obese animals. EC overexpression of wild-type and a cytosol-restricted mutant PFKFB3 decreased NO production due to increased eNOS-T495 phosphorylation. PFKFB3 also blunted Akt-S473 phosphorylation, reducing stimulus-dependent phosphorylation of S1177 and the activation of eNOS. Furthermore, PFKFB3 enhanced the activities of NOX1 and NOX5, which are major contributors to endothelial dysfunction. Prolonged exposure of ECs to high glucose or TNFα, which are hallmarks of T2D, leads to increased PFKFB3 expression. These results demonstrate a novel functional relationship between endothelial metabolism, ROS, and NO balance that may contribute to endothelial dysfunction in obesity.

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.

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.

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.

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.

Pneumolysin (PLY) is a bacterial pore forming toxin and primary virulence factor of Streptococcus pneumonia, a major cause of pneumonia. PLY binds cholesterol-rich domains of the endothelial cell (EC) plasma membrane resulting in pore assembly and increased intracellular (IC) Ca²⁺ levels that compromise endothelial barrier integrity. Caveolae are specialized plasmalemma microdomains of ECs enriched in cholesterol. We hypothesized that the abundance of cholesterol-rich domains in EC plasma membranes confers cellular susceptibility to PLY. Contrary to this hypothesis, we found increased PLY-induced IC Ca²⁺ following membrane cholesterol depletion. Caveolin-1 (Cav-1) is an essential structural protein of caveolae and its regulation by cholesterol levels suggested a possible role in EC barrier function. Indeed, Cav-1 and its scaffolding domain peptide protected the endothelial barrier from PLY-induced disruption. In loss of function experiments, Cav-1 was knocked-out using CRISPR-Cas9 or silenced in human lung microvascular ECs. Loss of Cav-1 significantly enhanced the ability of PLY to disrupt endothelial barrier integrity. Rescue experiments with re-expression of Cav-1 or its scaffolding domain peptide protected the EC barrier against PLY-induced barrier disruption. Dynamin-2 (DNM2) is known to regulate caveolar membrane endocytosis. Inhibition of endocytosis, with dynamin inhibitors or siDNM2 amplified PLY induced EC barrier dysfunction. These results suggest that Cav-1 protects the endothelial barrier against PLY by promoting endocytosis of damaged membrane, thus reducing calcium entry and PLY-dependent signaling.

Purinergic G-protein-coupled receptors are ancient and the most abundant group of G-protein-coupled receptors (GPCRs). The wide distribution of purinergic receptors in the cardiovascular system, together with the expression of multiple receptor subtypes in endothelial cells (ECs) and other vascular cells demonstrates the physiological importance of the purinergic signaling system in the regulation of the cardiovascular system. This review discusses the contribution of purinergic P2Y receptors to endothelial dysfunction (ED) in numerous cardiovascular diseases (CVDs). Endothelial dysfunction can be defined as a shift from a “calm” or non-activated state, characterized by low permeability, anti-thrombotic, and anti-inflammatory properties, to a “activated” state, characterized by vasoconstriction and increased permeability, pro-thrombotic, and pro-inflammatory properties. This state of ED is observed in many diseases, including atherosclerosis, diabetes, hypertension, metabolic syndrome, sepsis, and pulmonary hypertension. Herein, we review the recent advances in P2Y receptor physiology and emphasize some of their unique signaling features in pulmonary endothelial cells.

Obesity is a major and growing health problem in the US with over 93 million afflicted. Although, obesity itself is not considered a disease, it is a strong risk factor for cardiovascular disease, in large part due to an altered metabolic state. The relationship between metabolic dysfunction and impaired cardiovascular health remain incompletely understood. One of the earliest consequences of cardiovascular dysfunction in obesity is the loss of endothelial function and impaired nitric oxide (NO) signaling. NO is best known as the mediator of endothelium‐dependent relaxation, but it is also vital for maintaining blood vessel homeostasis through inhibition of leukocyte adhesion and smooth muscle cell proliferation. In blood vessels, NO is synthesized by endothelial nitric oxide synthase (eNOS) and dysregulation of eNOS has been shown to occur through changes in its expression and post‐translational phosphorylation. The biological actions of NO can be further compromised by rapid inactivation by reactive oxygen species (ROS) such as superoxide that are generated by NADPH oxidases (NOX) as well as numerous other enzyme systems. Our laboratory has previously shown that an imbalance between NO and superoxide‐generating pathways is responsible for the loss of endothelial function in obesity. However, the signals coordinating the loss of NO and increased superoxide with altered metabolism remain poorly defined. In our studies we have found that expression of the glycolytic enzyme 6‐phosphofructo‐2‐kinase/fructose‐2,6‐biphosphatase 3 (PFKFB3) is significantly increased in endothelial cells from obese animals. PFKFB3 is responsible for fructose‐2,6‐bisphosphate generation in vascular cells which is a potent allosteric activator of phosphofructokinase‐1 (PFK‐1), one of the rate‐limiting enzymes of glycolysis. We have also found that adenovirus‐mediated expression of PFKFB3 within the endothelium of mouse blood vessels is accompanied by the loss of endothelium‐dependent relaxation. In addition to its effects on blood vessels, overexpression of PFKFB3 in cultured cells resulted in decreased NO production as a result of increased eNOS phosphorylation on the well‐known inhibitory PKC phosphorylation site, threonine 495 (T495). Furthermore, PFKFB3 overexpression blunted Akt‐S473 phosphorylation, reducing stimulus‐dependent phosphorylation of eNOS at S1177. In addition to its negative effect on NO signaling overexpression of PFKFB3 increased the activity and mRNA level of NOX1, a major contributor to endothelial dysfunction in obesity. These results demonstrate a novel functional relationship between endothelial cell metabolism, nitric oxide and ROS production that may contribute to endothelial dysfunction in obesity. Support or Funding Information: 1R01HL147159‐01

Maintenance of the endothelial cell (EC) barrier is critical to vascular homeostasis and a loss of barrier integrity results in increased vascular permeability. While the mechanisms that govern increased EC permeability have been under intense investigation over the past several decades, the processes regulating the preservation/restoration of the EC barrier remain poorly understood. Herein we show that the extracellular purines, adenosine (Ado) and adenosine 5′‐[γ‐thio]‐triphosphate (ATPγS) can strengthen the barrier function of human lung microvascular EC (HLMVEC). This ability involves protein kinase A (PKA) activation and decreases in myosin light chain 20 (MLC20) phosphorylation secondary to the involvement of MLC phosphatase (MLCP). In contrast to Ado, ATPγS‐induced PKA activation is accompanied by a modest, but significant decrease in cyclic adenosine monophosphate (cAMP) levels supporting the existence of an unconventional cAMP‐independent pathway of PKA activation. Furthermore, ATPγS‐induced EC barrier strengthening does not involve the Rap guanine nucleotide exchange factor 3 (EPAC1) which is directly activated by cAMP but is instead dependent upon PKA‐anchor protein 2 (AKAP2) expression. We also found that AKAP2 can directly interact with the myosin phosphatase‐targeting protein MYPT1 and that depletion of AKAP2 abolished ATPγS‐induced increases in transendothelial electrical resistance. Ado‐induced strengthening of the HLMVEC barrier required the coordinated activation of PKA and EPAC1 in a cAMP‐dependent manner. In summary, ATPγS‐induced enhancement of the EC barrier is EPAC1‐independent and is instead mediated by activation of PKA which is then guided by AKAP2, in a cAMP‐independent mechanism, to activate MLCP which dephosphorylates MLC20 resulting in reduced EC contraction and preservation.

Microcystin-LR (MCY-LR) is a cyanobacterial toxin, a specific inhibitor of type 1 and 2A protein phosphatases (PP1 and PP2A) with significant impact on aquatic ecosystems. It has the potential to alter regulation of the plant cell cycle. The aim of this study was improved understanding of the mitotic alterations induced by cyanotoxin in Vicia faba, a model organism for plant cell biology studies. Vicia faba seedlings were treated over the long and short term with MCY-LR purified in our laboratory. Short-term treatments were performed on root meristems synchronized with hydroxylurea. Sections of lateral root tips were labelled for chromatin, phosphorylated histone H3 and β-tubulin via histochemical and immunohistochemical methods. Mitotic activity and the occurrence of mitotic alterations were detected and analysed by fluorescence microscopy. The phosphorylation state of histone H3 was studied by Western blotting. Long-term MCY-LR exposure of lateral root tip meristems increased the percentage of either early or late mitosis in a concentration-dependent manner. We observed hypercondensed chromosomes and altered sister chromatid segregation (lagging chromosomes) leading to the formation of micronuclei, accompanied by the formation of disrupted, multipolar and monopolar spindles, disrupted phragmoplasts and the hyperphosphorylation of histone H3 at Ser10. Short-term MCY-LR treatment of synchronized cells showed that PP1 and PP2A inhibition delayed the onset of anaphase at 1 µg mL⁻¹ MCY-LR, accelerated cell cycle at 10 µg mL⁻¹ MCY-LR and induced the formation of lagging chromosomes. In this case mitotic microtubule alterations were not detected, but histone H3 was hyperphosphorylated. MCY-LR delayed metaphase–anaphase transition. Consequently, it induced aberrant chromatid segregation and micronucleus formation that could be associated with both H3 hyperphosphorylation and altered microtubule organization. However, these two phenomena seemed to be independent. The toxin may be a useful tool in the study of plant cell cycle regulation.