Pyruvate kinase M2 (PKM2), a glycolytic enzyme, is required to maintain vascular barrier function

RATIONALE - Metabolic enzymes, like pyruvate kinase M2 (PKM2), play an essential role in altering endothelial cell (EC) phenotypes and behavior. Extensive research has elucidated the function of PKM2, a rate-limiting glycolytic enzyme, in the context of cancer cells and in activated immune cells, but its role in EC biology is only newly emerging. Recent findings show PKM2 acts as a key regulator of angiogenesis. Where exogenous circulating PKM2 induces EC cell proliferation leading to increased tumor angiogenesis and growth. Also, PKM2 deficient ECs exhibit decreased proliferation and migration. The relevance of PKM2 in modulating vascular barrier function is yet to be defined. OBJECTIVE -This study attempts to elucidate the role of PKM2 in regulating vascular barrier function. METHODS AND RESULTS - In vivo, EC specific deletion of PKM2 promotes increased vascular permeability in pulmonary capillary vessels and increased VEGF-induced acute vessel permeability in mouse dermal vessels. Similarly, in vitro, PKM2 deficient ECs exhibit decreased electrical resistance, disrupted VE-cadherin junctions and gap formations (illustrated via florescent VE-cadherin staining and phosphorylation of VE- cadherin protein at tyrosine residue Y658). Mechanistically, the deletion of PKM2 in ECs leads to increased angiopoietin-2 (Ang-2) expression, a well-known modulator of vascular permeability. Also, deletion of Ang-2 was sufficient to attenuate vascular leakage in PKM2 deficient endothelium, indicating that vascular leaky phenotype observed in PKM2 deficient endothelium is mediated by increased Ang-2 expression. CONCLUSIONS - PKM2, by modulating Ang-2 expression, plays a vital role in maintaining vascular barrier function.2019-07-11T00:00:00

Drug repurposing screen identifies Foxo1-dependent angiopoietin-2 regulation in sepsis

OBJECTIVE: The recent withdrawal of a targeted sepsis therapy has diminished pharmaceutical enthusiasm for developing novel drugs for the treatment of sepsis. Angiopoietin-2 is an endothelial-derived protein that potentiates vascular inflammation and leakage and may be involved in sepsis pathogenesis. We screened approved compounds for putative inhibitors of angiopoietin-2 production and investigated underlying molecular mechanisms. DESIGN: Laboratory and animal research plus prospective placebo-controlled randomized controlled trial (NCT00529139) and retrospective analysis (NCT00676897). SETTING: Research laboratories of Hannover Medical School and Harvard Medical School. PATIENTS: Septic patients/C57Bl/6 mice and human endothelial cells. INTERVENTIONS: Food and Drug Administration-approved library screening. MEASUREMENTS AND MAIN RESULTS: In a cell-based screen of more than 650 Food and Drug Administration-approved compounds, we identified multiple members of the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor drug class (referred to as statins) that suppressed angiopoietin-2. Simvastatin inhibited 3-hydroxy-3-methyl-glutaryl-CoA reductase, which in turn activated PI3K-kinase. Downstream of this signaling, PI3K-dependent phosphorylation of the transcription factor Foxo1 at key amino acids inhibited its ability to shuttle to the nucleus and bind cis-elements in the angiopoietin-2 promoter. In septic mice, transient inhibition of angiopoietin-2 expression by liposomal siRNA in vivo improved absolute survival by 50%. Simvastatin had a similar effect, but the combination of angiopoietin-2 siRNA and simvastatin showed no additive benefit. To verify the link between statins and angiopoietin-2 in humans, we performed a pilot matched case-control study and a small randomized placebo-controlled trial demonstrating beneficial effects on angiopoietin-2. CONCLUSIONS: 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors may operate through a novel Foxo1-angiopoietin-2 mechanism to suppress de novo production of angiopoietin-2 and thereby ameliorate manifestations of sepsis. Given angiopoietin-2's dual role as a biomarker and candidate disease mediator, early serum angiopoietin-2 measurement may serve as a stratification tool for future trials of drugs targeting vascular leakage

Endothelial pyruvate kinase M2 maintains vascular integrity

The M2 isoform of pyruvate kinase (PKM2) is highly expressed in most cancer cells, and has been studied extensively as a driver of oncogenic metabolism. In contrast, the role of PKM2 in nontransformed cells is little studied, and nearly nothing is known of its role, if any, in quiescent cells. We show here that endothelial cells express PKM2 almost exclusively over PKM1. In proliferating endothelial cells, PKM2 is required to suppress p53 and maintain cell cycle progression. In sharp contrast, PKM2 has a strikingly different role in quiescent endothelial cells, where inhibition of PKM2 leads to degeneration of tight junctions and barrier function. Mechanistically, PKM2 regulates barrier function independently of its canonical activity as a pyruvate kinase. Instead, PKM2 suppresses NF-kB and its downstream target, the vascular permeability factor angiopoietin 2. As a consequence, loss of endothelial cell PKM2 in vivo predisposes mice to VEGF-induced vascular leak, and to severe bacteremia and death in response to sepsis. Together, these data demonstrate new roles of PKM2 in quiescent cells, and highlight the need for caution in developing cancer therapies that target PKM2

A role of stochastic phenotype switching in generating mosaic endothelial cell heterogeneity

Previous studies have shown that biological noise may drive dynamic phenotypic mosaicism in isogenic unicellular organisms. However, there is no evidence for a similar mechanism operating in metazoans. Here we show that the endothelial-restricted gene, von Willebrand factor (VWF), is expressed in a mosaic pattern in the capillaries of many vascular beds and in the aorta. In capillaries, the mosaicism is dynamically regulated, with VWF switching between ON and OFF states during the lifetime of the animal. Clonal analysis of cultured endothelial cells reveals that dynamic mosaic heterogeneity is controlled by a low-barrier, noise-sensitive bistable switch that involves random transitions in the DNA methylation status of the VWF promoter. Finally, the hearts of VWF-null mice demonstrate an abnormal endothelial phenotype as well as cardiac dysfunction. Together, these findings suggest a novel stochastic phenotype switching strategy for adaptive homoeostasis in the adult vasculature