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.

Angiogenesis is a fundamental property of endothelium, yet not all endothelial cells display equivalent angiogenic responses; pulmonary microvascular endothelial cells undergo rapid angiogenesis when compared to endothelial cells isolated from conduit vessels. At present it is not clear how pulmonary microvascular endothelial cells fulfill the bioenergetic demands that are necessary to sustain such rapid blood vessel formation. We have previously established that pulmonary microvascular endothelial cells utilize aerobic glycolysis to generate ATP during growth, a process that requires the expression of lactate dehydrogenase A to convert pyruvate to lactate. Here, we test the hypothesis that lactate dehydrogenase A is required for pulmonary microvascular endothelial cells to sustain rapid angiogenesis. To test this hypothesis, Tet-On and Tet-Off conditional expression systems were developed in pulmonary microvascular endothelial cells, where doxycycline is utilized to induce lactate dehydrogenase A shRNA expression. Expression of LDH-A shRNA induced a time-dependent decrease in LDH-A protein, which corresponded with a decrease in glucose consumption from the media, lactate production and cell growth; re-expression of LDH-A rescued each of these parameters. LDH-A silencing greatly reduced network formation on Matrigel in vitro, and decreased blood vessel formation in Matrigel in vivo. These findings demonstrate that LDH-A is critically important for sustaining the rapid angiogenesis of pulmonary microvascular endothelial cells.

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Pulmonary microvascular endothelial cells (PMVECs) constitutively up‐regulate glycolytic enzymes necessary to generate ATP. One of these enzymes is lactate dehydrogenase (LDH), which converts pyruvate to lactate, causing lactic acidosis in proliferating cells. Since the role of LDH and lactate in endothelial pro‐proliferation is unknown, we tested whether lactate is a key contributor of rapid PMVEC proliferation. PMVEC growth curves were generated using standard culture conditions (10% serum with 22 mM glucose) in the presence of the LDH inhibitor, oxamate (4, 8 and 16 mM). Oxamate inhibited growth, glucose consumption and lactate production in a dose‐dependent manner, suggesting that conversion of pyruvate to lactate is essential for rapid proliferation. To determine whether glycolytic flux is necessary to sustain proliferation, galactose was substituted for glucose, since galactose is an ineffective glycolysis substrate. Galactose treatment reduced cellular ATP concentrations ?2‐fold, and inhibited growth, glucose consumption and lactate production. To examine whether lactate results in glycolytic‐independent cell growth, lactate was added to galactose‐rich media. Addition of exogenous lactate restored cellular ATP concentrations and partially rescued PMVEC proliferation. Thus, LDH contributes to the rapid PMVEC proliferation by controlling the rate of pyruvate conversion to lactate.

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