Point:Counterpoint: Chronic hypoxia-induced pulmonary hypertension does/does not lead to loss of pulmonary vasculature POINT: CHRONIC HYPOXIA-INDUCED PULMONARY HYPERTENSION DOES LEAD TO LOSS OF PULMONARY VASCULATURE

This debate focuses on whether, and to what extent, loss of small precapillary arteries is associated with the elevation in pulmonary artery pressure and resistance that accompanies chronic hypoxia-induced pulmonary hypertension. The issue has become the focus of much debate for a number of reasons. Papers from McLoughlin and his group (5, 6) have provided conclusive evidence that there is considerable angiogenesis associated with hypoxia-induced pulmonary hypertension. In addition, Rho kinase inhibitors normalize pulmonary pressure in chronic hypoxia presumably by a pure vasodilatory action (7). The fact that chronic hypoxia can stimulate neoangiogenesis does not negate the fact that chronic hypoxia can also result in loss of precapillary arteries. It is now generally accepted that loss of distal arteries in the clinical and experimental setting of pulmonary hypertension is the result of apoptosis of both endothelial cells and pericytes. This has been well documented in the monocrotaline model of pulmonary hypertension (24). Using this model, Stewart and colleagues (24) used fluorescent microbeads to document both breaks and abrupt termination of precapillary vessels, a feature associated with severe pulmonary hypertension. The authors went on to show how infusion of endothelial progenitor cells that synthesize endothelial nitric oxide synthase can reverse the pulmonary hypertension in association with rebuilding the distal vasculature that had been interrupted. In the clinical setting, a fulminant form of neoangiogenesis is associated with end-stage primary and secondary forms of pulmonary hypertension (4). This does not produce an effective increase in pulmonary flow through conduit channels, but rather represents structurally and functionally dysregulated vessels that resemble tumor vessels (4). These abnormal angiogenic channels are thought to arise because of the proliferation of “apoptosis-resistant” local endothelial cells or from the seeding of the lumen with circulating progenitor endothelial cells (20). A variety of pulmonary hypertension-inducing stimuli used in the experimental setting or related to clinical disease are associated with histological evidence of loss of distal pulmonary arteries, assessed either by platelet endothelial cell adhesion molecule (PECAM) staining of endothelial cells or by barium-gelatin infusion of the lungs. Experimental stimuli in addition to chronic hypoxia that induce loss of vessels in association with pulmonary hypertension have included injection of the toxin monocrotaline (23), monocrotaline and pneumonectomy (17), exposure to chronic hyperoxia (11, 22), and creation of aortopulmonary shunts (19). In the clinical setting, conditions associated with pulmonary hypertension and loss of arteries include congenital heart defects (18), lung developmental abnormalities (2), and idiopathic pulmonary hypertension (16). Improved resolution of current imaging techniques might, in the future, detect loss of precapillary arteries in association with pulmonary hypertension in the clinical setting. Loss of arteries reflecting loss of vascular reserve might be reflected in heightened pulmonary artery pressure and resistance with exercise or changes in pulmonary vascular impedance, which most accurately represents the total right ventricular afterload, including both steady and pulsatile right ventricular work requirements (21). Unfortunately, only the minority of clinical or experimental studies of chronic hypoxia-induced pulmonary hypertension report whether there is loss of precapillary vessels. One of the ways in which the number of precapillary vessels is precisely determined is by barium-gelatin infusion. This barium permits radiographic assessment of the circulation and the gelatin does not allow the contrast to pass into the capillary bed. Thus it is easy to count barium-filled peripheral arteries at alveolar duct and wall level on microscopic examination of lung tissue sections. Usually the number of precapillary arteries is recorded as the number of arteries relative to 100 alveoli or per squared millimeter. Calculating arteries per 100 alveoli makes the assumption that the alveoli are normal in number and calculating squared millimeter makes the assumption that the number and size of alveoli are normal. In addition to distensibility analysis (21), microCT (8) can be used to support loss of filling of distal arteries following exposure to chronic hypoxia using the barium-gelatin method or perfluorooctyl bromide (PFOB) as an intravascular X-ray contrast agent. With this method, isolated lungs harvested from mice are rinsed of blood and perfused with a physiological salt solution containing 5% bovine serum albumin while being ventilated with a 15% O2, 6% CO2, balance nitrogen mixture. Papavarine is added to the perfusate and recirculated prior to imaging to remove residual muscle tone. The perfusate is then replaced with PFOB, which is trapped at the precapillary level and only fills the arterial vasculature. High-resolution planar images are taken at an airway pressure of 6 mmHg for a range of intravascular pressures (between 6 and 17 mmHg). Alternatively, one can use vWF or PECAM staining of endothelium to landmark arteries accompanying alveolar ducts and alveolar walls down to a precapillary size of 20 m and to express those arteries relative to alveoli. This technique runs the risk of including venules in the assessment, but venules can generally be differentiated from arterioles since they are surrounded by loose connective tissue, they run in connective tissue septae in the lung, and they often have prominent branches. One of our recent studies has shown excellent correlation between the barium-gelatin and vWF immunostaining techniques to assess precapillary arteries (15). With these techniques, a reduction in the number of arteries relative to alveoli has been documented in rodents with chronic hypoxiainduced pulmonary hypertension in our laboratory (13, 14) and in that of others (3, 12). Studies using transgenic mice have taught us that there can be tremendous discrepancies between the hemodynamic assessments of pulmonary artery pressure and resistance and the remodeling response of the distal circulation in terms of muscularization of distal vessels, hypertrophy of more proximal arteries, and loss of arteries relative to alveoli. For example, in mice with overexpression of S100A4/Mts1, a baseline increase J Appl Physiol 103: 1449–1454, 2007; doi:10.1152/japplphysiol.00274.2007.