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POINT-COUNTERPOINT
1School of Medicine and Medical Science
Conway Institute
University College
Dublin, Ireland
e-mail: paul.mcloughlin{at}ucd.ie
2Department of Pharmacology and Center for Lung Biology
University of South Alabama
Exposure of native sea level dwellers to chronic hypoxia due to migration to high altitude leads to the development of increased pulmonary vascular resistance causing pulmonary hypertension. In some susceptible individuals this progressively worsens causing right ventricular failure and ultimately death. The increased pulmonary vascular resistance has previously been attributed to two structural changes in the pulmonary vascular bed: inward remodelling of the pulmonary arterioles leading to narrowing of the lumen and loss of pulmonary blood vessels, i.e., vascular rarefaction. It is the latter change that is the focus of this debate. While many groups have reported loss of vessels during exposure of rodents to chronic hypoxia (10, 11, 15, 16, 18, 25, 26, 29), several other groups could find no such loss (3, 5, 6, 14, 17, 19) and more recently some have provided evidence of new vessel formation in the pulmonary vasculature in response to sustained hypoxia (1, 12, 13, 22).
The evidence that hypoxia-induced pulmonary hypertension causes loss of vessels from the pulmonary circulation may be categorized under three broad headings: histological data, angiographic data, and functional data demonstrating the "fixed" nature of the increased pulmonary vascular resistance; we will consider each of these in turn. With histological techniques, the ability to detect loss of blood vessels in any tissue depends critically on the method used to identify them. A method commonly used in the pulmonary circulation has been to isolate the lung post mortem and then to perfuse a barium-gelatin mixture into the pulmonary artery at high pressure. As this mixture cools it solidifies within the arterial side of the circulation so that arterial vessels may be easily identified microscopically. Although intuitively attractive, this approach is fraught with difficulties, as the distance to which the barium-gelatin mix penetrates is a complex function of the vascular resistance, the rate of cooling of the mixture during infusion, and its viscosity. Increased resistance to flow of the mix, whether due to vasoconstriction or structural alterations of the vessels, will reduce the number of vessels filled and identified (6). Indeed it has been reported that, if the perfusion pressure used in chronically hypoxic lungs is elevated to compensate for their elevated vascular resistance, no evidence of vascular loss can be found (6). A further problem with the barium-gelatin method is that it only permits identification of arterial vessels while excluding the capillary and venous beds. These latter two segments are major sites of new vessel formation in the systemic circulation, suggesting that their exclusion when assessing the pulmonary circulation may be misleading (23). Thus vascular density data obtained using barium-gelatin injection must be interpreted with caution. Alternative ways of identifying pulmonary vessels include the use of elastin stains, immunostaining with endothelial cell markers, or the use of resin embedding, permitting thin sectioning and reliable morphometric identification. Interestingly, results obtained using these methods frequently do not show vessel loss (3, 12–14, 22).
Once tissue sections have been obtained, the extent of the vascular bed must then be quantified. Obtaining reliable three-dimensional data using two-dimensional sections is not as straightforward as it might at first appear (2, 9, 28). A commonly used approach has been to take a single transverse section of the left lung at the level of the hilum, to count the number of vessels and alveoli observed, and to compute the ratio of these two or, alternatively, to compute the number of vessels per unit area of the section; the resultant value has been loosely called "vessel density." The first problem with this approach is that the section is not representative of the lung as a whole. A second problem is that the number of intersections between a section and blood vessels is not a unique function of vessel length but also depends on the relative orientation of the plane of section and the vessels (2, 9, 28). Thus a single section (or multiple sections of a single fixed orientation) does not allow reliable estimation of vessel length. Perhaps most importantly, this method is not sensitive to the increases in lung volume caused by hypoxia (4, 10, 12–14, 24, 25). For example, vessel density as described above could remain unchanged if the lung enlarged and grew new vessels proportionately.
Use of stereological techniques allows unbiased quantitative analysis of the three-dimensional structure of the lung vasculature. Important aspects of the method are the use of systematic random sampling from throughout the lung, to ensure that the data obtained are representative of the whole organ, and quantification of changes in lung volume. This allows absolute quantities to be measured even in circumstances where the total lung volume changes (2, 9, 28). Use of stereology demonstrates new vessel formation in the pulmonary circulation in response to chronic hypoxia, not vessel loss (1, 12, 13). This finding is supported by previous work in which the pulmonary vascular volume, estimated by filling it with a solution containing tritiated albumin, was found to be increased in chronically hypoxic lungs (5).
Angiographic techniques form the second category of methods used to examine the structural changes in the pulmonary circulation following chronic hypoxia and have frequently been reported as showing a loss of pulmonary vessels. However, the problem is again that filling of the blood vessels is influenced by pulmonary vascular resistance and is therefore not a reliable method for identifying vessels. For example, it has been shown that the extent of the vascular bed revealed by such methods critically depends on the perfusion pressure (6).
The final category of evidence that is used to support the view that structural changes underlie hypoxic pulmonary hypertension is functional in nature. Once chronic hypoxic pulmonary hypertension has become established, abrupt return to normoxic conditions does not cause an immediate substantial fall in pulmonary arterial pressure (7, 8). Moreover, most vasodilator agents only produce small acute falls in pulmonary arterial pressure and vascular resistance remains substantially elevated above control values. This has been interpreted as showing that ongoing hypoxic vasoconstriction is not a significant contributor to the established pulmonary hypertension and that "fixed" structural changes, including inward remodelling and the loss of pulmonary vessels, must be the dominant mechanism. However, more recently it has been appreciated that the RhoA-Rho kinase pathway, through its effect on myosin light chain phosphatase activity and thus sustained vascular smooth muscle contraction, has a very important role in the chronically hypoxic lung. This pathway is upregulated in the pulmonary vascular smooth muscle in hypoxic pulmonary hypertension, and acute administration of inhibitors of Rho kinase to chronically hypoxic hypertensive lungs immediately reduces pulmonary vascular resistance to values that are close to normal (13, 20, 21). Such an immediate return of pulmonary vascular resistance to near control values is not compatible with a major role for structural changes, including either hypertrophic luminal encroachment or vessel loss, as the basis for chronic hypoxic pulmonary hypertension. A recent editorial has addressed the likely role of sustained vasoconstriction in the apparent structural inward remodelling of hypoxia-induced hypertensive rat pulmonary arteries (27). Whether the pulmonary arterial wall thickening has an important negative impact on vascular distensibility in the chronically hypoxic lung remains to be determined.
In summary, we conclude that structural data obtained using rigorous stereological techniques and functional evidence obtained with recently developed inhibitors of the RhoA-Rho kinase pathway demonstrate that chronic alveolar hypoxia, acting in the absence of any lung disease, does not cause loss of pulmonary vessels.
GRANTS
P. McLoughlin is funded by program grants from the Health Research Board (Ireland) and PRTLI Higher Education Authority (Ireland). I. McMurtry receives funding from the National Heart, Lung, and Blood Institute (HL-14985).
REFERENCES
protects mice against pulmonary hypertension and right ventricular dysfunction during prolonged hypoxia. J Clin Invest 111: 1519–1527, 2004.[CrossRef][Web of Science]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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 M. O. Leonard, K. Howell, S. F. Madden, C. M. Costello, D. G. Higgins, C. T. Taylor, and P. McLoughlin Hypoxia Selectively Activates the CREB Family of Transcription Factors in the In Vivo Lung Am. J. Respir. Crit. Care Med., November 1, 2008; 178(9): 977 - 983. [Abstract] [Full Text] [PDF]
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