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POINT-COUNTERPOINT
1Department of Pediatrics
Stanford University School of Medicine
e-mail: marlener{at}stanford.edu
2Department of Biomedical Engineering
University of Wisconsin-Madison
Zablocki Veterans Affairs Medical Center
3Department of Medicine-PCC
Medical College of Wisconsin
Milwaukee, Wisconsin
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 µm 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 hypoxia-induced 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 in pulmonary artery pressure is further augmented by chronic hypoxia relative to controls, but we were unable to identify an increase in the muscularization of distal vessels, in the loss of distal vessels landmarked as precapillary, or in the wall thickness of normally muscular arteries that would explain this discrepancy. We did, however, document marked changes in the elastin matrix (14, 15) that might have influenced the distensibility characteristics in the pulmonary circulation (9, 10).
We observed that patchy deletion of BMPR1a in smooth muscle cells and others have reported that haploinsufficiency of BMPRII results in equivalent pulmonary artery pressures found in wild-type mice exposed to chronic hypoxia, but less structural remodeling of the distal circulation (1). New studies are considering the contribution of the extracellular matrix, where an increase in aberrantly distributed elastin and collagen could be associated with reduced compliance (9, 10) and thus increased impedance even when resistance is unchanged.
So, the following might summarize what we believe is the basis for the difference of opinion regarding hypoxia-mediated loss of distal arteries.
First, pulmonary hypertension is reversed with Rho kinase inhibitors. However, this does not negate the loss of vessels, only that under basal conditions, the loss of distal arteries does not impair resting steady hemodynamics of the pulmonary circulation.
Second, stereology shows increased angiogenesis and increased capillary length in hypoxia. This is well documented but does not tell us about aberrant or "lost" connections between the precapillary and capillary circulation.
Third, loss of arteries is not always seen in hypoxia. Certain methodologies like barium-gelatin injection are designed to facilitate assessment of the distal precapillary pulmonary vasculature, but this method can be technically challenging particularly in murine lungs. However, PECAM or vWF immunostaining validates the loss of vessels when used in the same series of experiments and this should also be possible with microCT with PFOB.
Fourth, certain murine species may not show loss of arteries. This may be true despite the fact that other features of remodeling of the pulmonary circulation are observed. Also, we need to look beyond the vascular changes we have typically observed in chronic hypoxia-induced pulmonary hypertension into those that affect the total right ventricular afterload, including both steady and pulsatile right ventricular work.
REFERENCES
This article has been cited by other articles:
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  K. Howell, C. M. Costello, M. Sands, I. Dooley, and P. McLoughlin L-Arginine promotes angiogenesis in the chronically hypoxic lung: a novel mechanism ameliorating pulmonary hypertension Am J Physiol Lung Cell Mol Physiol, June 1, 2009; 296(6): L1042 - L1050. [Abstract] [Full Text] [PDF]
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 E. Daley, C. Emson, C. Guignabert, R. de Waal Malefyt, J. Louten, V. P. Kurup, C. Hogaboam, L. Taraseviciene-Stewart, N. F. Voelkel, M. Rabinovitch, et al. Pulmonary arterial remodeling induced by a Th2 immune response J. Exp. Med., February 18, 2008; 205(2): 361 - 372. [Abstract] [Full Text] [PDF]
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