Troy Stevens

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.

Rationale:: Canonical transient receptor potential 4 (TRPC4) contributes to the molecular composition of a channel encoding for a calcium selective store-operated current, I _SOC , whereas Orai1 critically comprises a channel encoding for the highly selective calcium release activated calcium current, I _CRAC . However, Orai1 may interact with TRPC proteins and influence their activation and permeation characteristics. Endothelium expresses both TRPC4 and Orai1, and it remains unclear as to whether Orai1 interacts with TRPC4 and contributes to calcium permeation through the TPRC4 channel. Objective:: We tested the hypothesis that Orai1 interacts with TRPC4 and contributes to the channel's selective calcium permeation important for endothelial barrier function. Methods and Results:: A novel method to purify the endogenous TRPC4 channel and probe for functional interactions was developed, using TRPC4 binding to protein 4.1 as bait. Isolated channel complexes were conjugated to anti-TRPC protein antibodies labeled with cy3-cy5 pairs. Förster Resonance Energy Transfer among labeled subunits revealed the endogenous protein alignment. One TRPC1 and at least 2 TRPC4 subunits constituted the endogenous channel (TRPC1/4). Orai1 interacted with TRPC4. Conditional Orai1 knockdown reduced the probability for TRPC1/4 channel activation and converted it from a calcium-selective to a nonselective channel, an effect that was rescued on Orai1 reexpression. Loss of Orai1 improved endothelial cell barrier function. Conclusion:: Orai1 interacts with TRPC4 in the endogenous channel complex, where it controls TRPC1/4 activation and channel permeation characteristics, including calcium selectivity, important for control of endothelial cell barrier function.

Pain, redness, heat, and swelling are hallmarks of inflammation that were recognized as early as the first century AD. Despite these early observations, the mechanisms responsible for swelling, in particular, remained an enigma for nearly two millennia. Only in the past century have scientists and physicians gained an appreciation for the role that vascular endothelium plays in controlling the exudation that is responsible for swelling. One of these mechanisms is the formation of transient gaps between adjacent endothelial cell borders. Inflammatory mediators act on endothelium to reorganize the cytoskeleton, decrease the strength of proteins that connect cells together, and induce transient gaps between endothelial cells. These gaps form a paracellular route responsible for exudation. The discovery that interendothelial cell gaps are causally linked to exudation began in the 1960s and was accompanied by significant controversy. Today, the role of gap formation in tissue edema is accepted by many, and significant scientific effort is dedicated toward developing therapeutic strategies that will prevent or reverse the endothelial cell gaps that are present during the course of inflammatory illness. Given the importance of this field in endothelial cell biology and inflammatory disease, this focused review catalogs key historical advances that contributed to our modern-day understanding of the cell biology of interendothelial gap formation.

In recent years there has been an increasing appreciation of the functional heterogeneity that exists between extraalveolar and alveolar endothelial cells. One of the most striking features of pulmonary microvascular endothelial cells is that they possess a highly impermeable barrier with respect to pulmonary artery or vein endothelial cells. This cellular feature is observed in culture and in the intact microcirculation, prompting a reevaluation of the key physiological principles that control permeability and the fate of fluid (or exudate) once it leaves the circulation. Pulmonary microvascular endothelial cells express calcium channels not found in extraalveolar endothelial cells, including the vanilloid family transient receptor potential 4 channel and the α1G T-type calcium channel. Whereas activation of the TRPV4 channel causes alveolar flooding, activation of the α1G T-type calcium channel promotes P-selectin surface translocation, events specific to the microcirculation. Although endothelium is an attractive therapeutic target in acute lung injury and other vascular disorders, the growing awareness of pulmonary endothelial cell heterogeneity increasingly suggests that a panendothelial cell approach is suboptimal. Rather, development of novel therapeutics based upon anatomically restricted expression of molecular signatures may be developed to better combat vascular disease.

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.

Objective: Lung inflammation causes perivascular fluid cuffs to form around extra-alveolar blood vessels; however, the physiologic consequences of such cuffs remain poorly understood. Herein, we tested the hypothesis that perivascular fluid cuffs, without concomitant alveolar edema, are sufficient to decrease lung compliance. Design: Prospective, randomized, controlled study. Setting: Research laboratory. Subjects: One hundred twenty male CD40 rats. Interventions: To test this hypothesis, the plant alkaloid thapsigargin was used to activate store-operated calcium entry and increase cytosolic calcium in endothelium. Thapsigargin was infused into a central venous catheter of intact, sedated, and mechanically ventilated rats. Measurements: Static and dynamic lung mechanics and hemodynamics were measured continuously. Main Results: Thapsigargin produced perivascular fluid cuffs along extra-alveolar vessels but did not cause alveolar flooding or blood gas abnormalities. Lung compliance dose-dependently decreased after thapsigargin infusion, attributable to an increase in tissue resistance that was attributed to increased tissue damping and tissue elastance. Airway resistance was not changed. Neither central venous pressure nor left ventricular end diastolic pressure was altered by thapsigargin. Heart rate did not change, although thapsigargin decreased left ventricular systolic function sufficient to reduce cardiac output by 50%. Infusion of the type 4 phosphodiesterase inhibitor, rolipram, prevented thapsigargin from inducing perivascular cuffs and decreasing lung compliance. Rolipram also normalized pressure over time and corrected the deficit in cardiac output. Conclusions: Our findings resolve for the first time that perivascular cuff formation negatively impacts mechanical coupling between the bronchovascular bundle and the lung parenchyma, decreasing lung compliance without impacting central venous pressure.

Quiescent pulmonary endothelium establishes an antithrombotic, anti-inflammatory surface that promotes blood flow. However, the endothelium rapidly responds to injury and inflammation by promoting thrombosis and enabling the directed transmigration of inflammatory cells, such as neutrophils, into the alveolar airspace. Although the endothelial cell signals responsible for establishing a prothrombotic surface are distinct from those responsible for recognizing circulating neutrophils, these processes are highly interrelated. Von Willebrand factor (VWF)-stimulated secretion plays an important role in thrombus formation, and P-selectin surface expression plays a key role in neutrophil binding necessary for transmigration. Both VWF and P-selectin are located within Weibel-Palade bodies in pulmonary arteries and arterioles, yet Weibel-Palade bodies are absent in capillaries. Despite the absence of the Weibel-Palade bodies, pulmonary capillaries express both VWF and P-selectin. The physiological and pathophysiological significance of these observations is unclear. In this review, we address some anatomical and physiological features that distinguish pulmonary artery, capillary, and vein endothelium. In addition, we review our current understanding regarding the stimulated secretion of VWF and P-selectin in pulmonary artery and capillary endothelium. This information is considered in the context of vasculitis and pneumonia, two pathophysiological processes to which the stimulated secretion of VWF and P-selectin contribute.

Cells of diverse origin utilize shifts in cytosolic calcium concentrations as intracellular signals to elicit physiological responses. In endothelium, inflammatory first messengers increase cytosolic calcium as a signal to disrupt cell-cell borders and produce inter-cellular gaps. Calcium influx across the plasma membrane is required to initiate barrier disruption, although the calcium entry mechanism responsible for this effect remains poorly understood. This chapter highlights recent efforts to define the molecular anatomy of the ion channel responsible for triggering endothelial cell gap formation. Resolving the identity and function of this calcium channel will pave the way for new anti-inflammatory therapeutic targets.

Soluble adenylyl cyclase toxins, such as Pseudomonas aeruginosa exoY, generate a cAMP pool that retracts cell borders. However, the cytoskeletal basis by which this cAMP signal retracts cell borders is not known. We sought to determine whether activation of chimeric, soluble adenylyl cyclase I/II (sACI/II) reorganizes either microtubules or peripheral actin. Endothelial cells were stably transfected with either green fluorescent protein-labeled α-tubulin or β-actin, and then infected with adenovirus to express sACI/II. Forskolin, which stimulates both the endogenously expressed transmembrane adenylyl cyclases and sACI/II, induced cell retraction accompanied by the reorganization of peripheral microtubules. However, cortical filamentous-actin (f-actin) did not reorganize into stress fibers, and myosin light-chain-20 phosphorylation was decreased. Isoproterenol, which activates endogenous adenylyl cyclases but does not activate sACI/II, did not induce endothelial cell gaps and did not influence microtubule or f-actin architecture. Thus, sACI/II generates a cAMP signal that reorganizes microtubules and induces cell retraction, without inducing f-actin stress fibers. These findings illustrate that endothelial cell gap formation can proceed without f-actin stress fiber formation, and provide mechanistic insight how bacterial adenylyl cyclase toxins reorganize the cytoskeleton to induce cell rounding.