Pulmonary microvascular endothelial cells (PMVECs) 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 (LDH-A) converts pyruvate to lactate necessary to sustain rapid flux through glycolysis. We therefore tested the hypothesis that PMVECs express LDH-A necessary to utilize aerobic glycolysis and support their growth and their neo-angiogenic capacity. We first characterized the glycolytic phenotype of PMVECs, as compared to the more oxidative metabolism of pulmonary artery endothelial cells (PAECs). PMVECs consumed glucose, converted glucose into lactate, and acidified the media. In contrast, slow growing PAECs minimally consumed glucose and did not develop a lactic acidosis throughout the growth curve. Oxygen consumption was two-fold higher in PAECs than in PMVECs, yet total cellular ATP concentrations were two-fold higher in PMVECs. Glucose transporter-1 (GLUT-1), Hexokinase-2 (HK-2), and LDH-A were all upregulated in PMVECs compared with their macrovascular counterparts. Inhibiting LDH-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 LDH-A.

To discern whether LDH-A activity is a determinant of neo-angiogenic capacity, we developed Tet-On and Tet-Off expression systems to conditionally control shRNA expression. In the Tet-On system, doxycycline induced LDH-A shRNA, which reduced LDH-A protein. In the Tet-Off system, cells were grown in doxycycline to suppress LDH-A shRNA; doxycycline retrieval induced shRNA leading to a decrease in LDH-A expression. Using these systems, LDH-A expression was shown to be required for PMVEC network formation in Matrigel in vitro, and for blood vessel formation in Matrigel in vivo. These findings reveal the importance of aerobic glycolysis, in particular LDH-A expression, in the pro-angiogenic phenotype of PMVECs.