Pulmonary arterial hypertension (PAH) is a progressive disease of the lung vasculature that involves the loss of endothelial function together with inappropriate smooth muscle cell growth, inflammation, and fibrosis. These changes underlie a progressive remodeling of blood vessels that alters flow and increases pulmonary blood pressure. Elevated pressures in the pulmonary artery imparts a chronic stress on the right ventricle which undergoes compensatory hypertrophy but eventually fails. How PAH develops remains incompletely understood and evidence for the altered production of reactive oxygen and nitrogen species (ROS, RNS respectively) in the pulmonary circulation has been well documented. There are many different types of ROS and RNS, multiple sources, and collective actions and interactions. This review summarizes past and current knowledge of the sources of ROS and RNS and how they may contribute to the loss of endothelial function and changes in smooth muscle proliferation in the pulmonary circulation.

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.

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.

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

Background: Streptococcus pneumonia is the primary etiological agent of community-acquired pneumonia (CAP). Pneumococci promote severe lung injury through the release of virulence factors, including pneumolysin (PLY). Obesity/diabetes increases pneumonia-associated mortality, but the mechanisms remain elusive. We found that obese db/db mice have increased pulmonary barrier disruption to PLY. Previously we showed that upregulation of NOX1 in endothelial cells (EC) of db/db mice drives endothelial dysfunction, but a role for NOX1 in PLY-induced lung injury, especially in diabetic conditions, has not yet been described. Results: Increased NOX1 in lung ECs dose-dependently increased superoxide and EC barrier disruption (p < 0.05). Even at low activity levels, NOX1 greatly potentiated PLY-induced EC barrier disruption, whereas loss of NOX1 activity, either pharmacological or genetic, reduced barrier disruption (p < 0.05). Blockade of calcium entry protected the EC barrier from combined PLY and NOX1, indicating a key role for calcium. Hyperglycemia amplified PLY-enduced EC barrier disruption and intracellular calcium and these effects were mitigated by NOX1 inhibition and silencing (p < 0.05). NOX1-enhanced calcium entry was reduced by knockout of calcium sensor STIM1, and PLY-induced barrier disruption was reduced by STIM1 inhibition. Levels of STIM1, Orai1, TRPV4, or TRPC4 were unchanged by HG, but TRPC1 significantly increased (p < 0.05). NOX1 and HG promoted increased STIM1 and TRPC1 binding, and silencing TRPC1 ameliorated PLY-induced barrier disruption (p < 0.05). Increased calcium promoted mitochondrial permeability transition pore (MPTP) opening and PPIF inhibition protected EC barrier function (p < 0.05). Conclusions: These results suggest that elevated glucose levels in obesity primes EC barrier disruption by amplifying PLY-induced calcium influx via a novel NOX1, STIM1, TRPC1 and MPTP signaling axis.