KD025, a ROCK2 Specific Inhibitor, Increases Oxidative Phosphorylation and Inhibits Aerobic Glycolysis, Intracellular pH, Migration and Network Formation in Pulmonary Microvascular Endothelial Cells in a ROCK2 Independent Manner

Pulmonary microvascular endothelial cells (PMVECs) display a rapid angioproliferative phenotype, essential for maintaining homeostasis in steady-state and promoting vascular repair after injury. Although it has long been established that endothelial cytosolic Ca2+ ([Ca2+]i) transients are required for proliferation and angiogenesis, mechanisms underlying such regulation and the transmembrane channels mediating the relevant [Ca2+]i transients remain incompletely understood. In the present study, the functional role of the microvascular endothelial site-specific α1G T-type Ca2+ channel in angiogenesis was examined. PMVECs intrinsically possess an in vitro angiogenic “network formation” capacity. Depleting extracellular Ca2+ abolishes network formation, whereas blockade of vascular endothelial growth factor receptor or nitric oxide synthase has little or no effect, suggesting that the network formation is a [Ca2+]i-dependent process. Blockade of the T-type Ca2+ channel or silencing of α1G, the only voltage-gated Ca2+ channel subtype expressed in PMVECs, disrupts network formation. In contrast, blockade of canonical transient receptor potential (TRP) isoform 4 or TRP vanilloid 4, two other Ca2+ permeable channels expressed in PMVECs, has no effect on network formation. T-type Ca2+ channel blockade also reduces proliferation, cell-matrix adhesion, and migration, three major components of angiogenesis in PMVECs. An in vivo study demonstrated that the mice lacking α1G exhibited a profoundly impaired postinjury cell proliferation in the lungs following lipopolysaccharide challenge. Mechanistically, T-type Ca2+ channel blockade reduces Akt phosphorylation in a dose-dependent manner. Blockade of Akt or its upstream activator, phosphatidylinositol-3-kinase (PI3K), also impairs network formation. Altogether, these findings suggest a novel functional role for the α1G T-type Ca2+ channel to promote the cell’s angiogenic potential via a PI3K-Akt signaling pathway.

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Extrinsic acidosis suppresses glycolysis and migration while increasing network formation in pulmonary microvascular endothelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00544.2018 ◽

2019 ◽

Vol 317 (2) ◽

pp. L188-L201 ◽

Cited By ~ 4

Author(s):

Ji Young Lee ◽

Mher Onanyan ◽

Ian Garrison ◽

Roderica White ◽

Maura Crook ◽

...

Keyword(s):

Endothelial Cells ◽

Network Formation ◽

Segment Length ◽

Microvascular Endothelial Cells ◽

Carbonic Anhydrases ◽

Pulmonary Microvascular Endothelial Cells ◽

Ca Ix ◽

Microvascular Endothelial ◽

And Migration ◽

The Impact

Acidosis is common among critically ill patients, but current approaches to correct pH do not improve disease outcomes. During systemic acidosis, cells are either passively exposed to extracellular acidosis that other cells have generated (extrinsic acidosis) or they are exposed to acid that they generate and export into the extracellular space (intrinsic acidosis). Although endothelial repair following intrinsic acidosis has been studied, the impact of extrinsic acidosis on migration and angiogenesis is unclear. We hypothesized that extrinsic acidosis inhibits metabolism and migration but promotes capillary-like network formation in pulmonary microvascular endothelial cells (PMVECs). Extrinsic acidosis was modeled by titrating media pH. Two types of intrinsic acidosis were compared, including increasing cellular metabolism by chemically inhibiting carbonic anhydrases (CAs) IX and XII (SLC-0111) and with hypoxia. PMVECs maintained baseline intracellular pH for 24 h with both extrinsic and intrinsic acidosis. Whole cell CA IX protein expression was decreased by extrinsic acidosis but not affected by hypoxia. When extracellular pH was equally acidic, extrinsic acidosis suppressed glycolysis, whereas intrinsic acidosis did not. Extrinsic acidosis suppressed migration, but increased Matrigel network master junction and total segment length. CRISPR-Cas9 CA IX knockout PMVECs revealed an independent role of CA IX in promoting glycolysis, as loss of CA IX alone was accompanied by decreased hexokinase I and pyruvate dehydrogenase E1α expression and decreasing migration. 2-deoxy-d-glucose had no effect on migration but profoundly inhibited network formation and increased N-cadherin expression. Thus, we report that while extrinsic acidosis suppresses endothelial glycolysis and migration, it promotes network formation.

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Critical role for lactate dehydrogenase A in aerobic glycolysis that sustains pulmonary microvascular endothelial cell proliferation

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00274.2009 ◽

2010 ◽

Vol 299 (4) ◽

pp. L513-L522 ◽

Cited By ~ 89

Author(s):

Glenda Parra-Bonilla ◽

Diego F. Alvarez ◽

Abu-Bakr Al-Mehdi ◽

Mikhail Alexeyev ◽

Troy Stevens

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Lactate Dehydrogenase ◽

Lactic Acidosis ◽

Aerobic Glycolysis ◽

Microvascular Endothelial Cells ◽

Microvascular Endothelial Cell ◽

Pulmonary Microvascular Endothelial Cells ◽

Pulmonary Microvascular Endothelial Cell ◽

Microvascular Endothelial

Pulmonary microvascular endothelial cells possess both highly proliferative and angiogenic capacities, yet it is unclear how these cells sustain the metabolic requirements essential for such growth. Rapidly proliferating cells rely on aerobic glycolysis to sustain growth, which is characterized by glucose consumption, glucose fermentation to lactate, and lactic acidosis, all in the presence of sufficient oxygen concentrations. Lactate dehydrogenase A converts pyruvate to lactate necessary to sustain rapid flux through glycolysis. We therefore tested the hypothesis that pulmonary microvascular endothelial cells express lactate dehydrogenase A necessary to utilize aerobic glycolysis and support their growth. Pulmonary microvascular endothelial cell (PMVEC) growth curves were conducted over a 7-day period. PMVECs consumed glucose, converted glucose into lactate, and acidified the media. Restricting extracellular glucose abolished the lactic acidosis and reduced PMVEC growth, as did replacing glucose with galactose. In contrast, slow-growing pulmonary artery endothelial cells (PAECs) minimally consumed glucose and did not develop a lactic acidosis throughout the growth curve. Oxygen consumption was twofold higher in PAECs than in PMVECs, yet total cellular ATP concentrations were twofold higher in PMVECs. Glucose transporter 1, hexokinase-2, and lactate dehydrogenase A were all upregulated in PMVECs compared with their macrovascular counterparts. Inhibiting lactate dehydrogenase A activity and expression prevented lactic acidosis and reduced PMVEC growth. Thus PMVECs utilize aerobic glycolysis to sustain their rapid growth rates, which is dependent on lactate dehydrogenase A.

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CaMK4 Is a Downstream Effector of the α1G T-type Calcium Channel to Determine the Angiogenic Potential of Pulmonary Microvascular Endothelial Cells

AJP Cell Physiology ◽

10.1152/ajpcell.00216.2021 ◽

2021 ◽

Author(s):

Zhen Zheng ◽

Xuelin Wang ◽

Yuxia Wang ◽

Judy A.C. King ◽

Peilin Xie ◽

...

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Network Formation ◽

Microvascular Endothelial Cells ◽

Angiogenic Potential ◽

Akt Phosphorylation ◽

Pulmonary Microvascular Endothelial Cells ◽

Downstream Effector ◽

Phosphorylation Level ◽

Microvascular Endothelial

Pulmonary microvascular endothelial cells (PMVECs) uniquely express an α1G-subtype of voltage-gated T-type Ca2+ channel. We have previously revealed that the α1G channel functions as a background Ca2+ entry pathway that is critical for the cell proliferation, migration, and angiogenic potential of PMVECs, a novel function attributed to the coupling between α1G-mediate Ca2+ entry and constitutive Akt phosphorylation and activation. Despite this significance, mechanism(s) that link the α1G-mediated Ca2+ entry to Akt phosphorylation remain incompletely understood. In the present study, we demonstrate that Ca2+/calmodulin-dependent protein kinase (CaMK) 4 serves as a downstream effector of the α1G-mediated Ca2+ entry to promote the angiogenic potential of PMVECs. Notably, CaMK2 and CaMK4 are both expressed in PMVECs. Pharmacological blockade or genetic knockdown of the α1G channel led to a significant reduction in the phosphorylation level of CaMK4 but not the phosphorylation level of CaMK2. Pharmacological inhibition as well as genetic knockdown of CaMK4 significantly decreased cell proliferation, migration, and network formation capacity in PMVECs. However, CaMK4 inhibition or knockdown did not alter Akt phosphorylation status in PMVECs, indicating that α1G/Ca2+/CaMK4 is independent of the α1G/Ca2+/Akt pathway in sustaining the cells' angiogenic potential. Altogether, these findings suggest a novel α1G-CaMK4 signaling complex that regulates the Ca2+-dominated angiogenic potential in PMVECs.

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Hypoxia-induced increase in NO production and eNOS expression by murine pulmonary microvascular endothelial cells

Pneumologie ◽

10.1055/s-2007-967250 ◽

2007 ◽

Vol 61 (01) ◽

Author(s):

R Paddenberg ◽

U Pfeil ◽

A Goldenberg ◽

P Faulhammer ◽

W Kummer

Keyword(s):

Endothelial Cells ◽

Microvascular Endothelial Cells ◽

No Production ◽

Enos Expression ◽

Pulmonary Microvascular Endothelial Cells ◽

Microvascular Endothelial

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Angiotensin I-converting enzyme (ACE) Expression in HPMEC (human pulmonary microvascular endothelial cells) nach LPS-Stimulation

Pneumologie ◽

10.1055/s-2007-973279 ◽

2007 ◽

Vol 61 (S 1) ◽

Author(s):

AM Müller ◽

I Hermanns ◽

F Franke ◽

C Kirkpatrick

Keyword(s):

Endothelial Cells ◽

Microvascular Endothelial Cells ◽

Converting Enzyme ◽

Angiotensin I ◽

Pulmonary Microvascular Endothelial Cells ◽

Angiotensin I Converting Enzyme ◽

Microvascular Endothelial

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USP18 mitigates lipopolysaccharide-induced oxidative stress and inflammation in human pulmonary microvascular endothelial cells through the TLR4/NF-κB/ROS signaling

Toxicology in Vitro ◽

10.1016/j.tiv.2021.105181 ◽

2021 ◽

pp. 105181

Author(s):

Zeyu Jiang ◽

Jiang Shen ◽

Jie Ding ◽

Yan Yuan ◽

Lulu Gao ◽

...

Keyword(s):

Oxidative Stress ◽

Endothelial Cells ◽

Microvascular Endothelial Cells ◽

Pulmonary Microvascular Endothelial Cells ◽

Ros Signaling ◽

Microvascular Endothelial

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Proproliferative phenotype of pulmonary microvascular endothelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00304.2006 ◽

2007 ◽

Vol 292 (3) ◽

pp. L671-L677 ◽

Cited By ~ 17

Author(s):

Victor Solodushko ◽

Brian Fouty

Keyword(s):

Endothelial Cells ◽

Barrier Function ◽

Pulmonary Arteries ◽

D1 Protein ◽

Coagulation Cascade ◽

Microvascular Endothelial Cells ◽

Significant Heterogeneity ◽

Pulmonary Microvascular Endothelial Cells ◽

Microvascular Endothelial ◽

Pulmonary Microcirculation

Endothelial cells perform a number of important functions including release of vasodilators, control of the coagulation cascade, and restriction of solutes and fluid from the extravascular space. Regulation of fluid balance is of particular importance in the microcirculation of the lung where the loss of endothelial barrier function can lead to alveolar flooding and life-threatening hypoxemia. Significant heterogeneity exists between endothelial cells lining the microcirculation and cells from larger pulmonary arteries, however, and these differences may be relevant in restoring barrier function following vascular injury. Using well-defined populations of rat endothelial cells harvested from the pulmonary microcirculation [pulmonary microvascular endothelial cells (PMVEC)] and from larger pulmonary arteries [pulmonary artery endothelial cells (PAEC)], we compared their growth characteristics in low serum conditions. Withdrawal of serum inhibited proliferation and induced G0/G1 arrest in PAEC, whereas PMVEC failed to undergo G0/G1 arrest and continued to proliferate. Consistent with this observation, PMVEC had an increased cdk4 and cdk2 kinase activity with hyperphosphorylated (inactive) retinoblastoma (Rb) relative to PAEC as well as a threefold increase in cyclin D1 protein levels; overexpression of the cdk inhibitors p21Cip1/Waf1 and p27Kip1 induced G0/G1 arrest. While serum withdrawal failed to induce G0/G1 arrest in nonconfluent PMVEC, confluence was associated with hypophosphorylated Rb and growth arrest; loss of confluence led to resumption of growth. These data suggest that nonconfluent PMVEC continue to proliferate independently of growth factors. This proliferative characteristic may be important in restoring confluence (and barrier function) in the pulmonary microcirculation following endothelial injury.

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