Structural basis of hypoxic pulmonary hypertension: the modifying effect of chronic hypercapnia

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1 Exp Physiol 89.1 pp Experimental Physiology Hot Topic Review Festschrift for R. G. O Regan Sensing and adaptation to alterations in respiratory gases: oxygen and carbon dioxide Structural basis of hypoxic pulmonary hypertension: the modifying effect of chronic hypercapnia Katherine Howell, Henry Ooi, Rob Preston and Paul McLoughlin Department of Human Anatomy and Physiology, Conway Institute of Biomolecular and Biomedical Research and Dublin Molecular Medicine Centre, University College Dublin, Ireland Exposure to chronic hypoxia causes pulmonary hypertension and pulmonary vascular remodelling. In chronic lung disease, chronic hypercapnia frequently coexists with hypoxia and is associated with worsening of pulmonary hypertension. It is generally stated that pulmonary hypertension in these conditions is secondary to hypoxic vascular remodelling and that hypercapnia augments this remodelling thus worsening the hypertension. We review recent evidence which shows that although chronic hypoxia causes thickening of the walls of pulmonary arterioles, these changes do not lead to structural narrowing of the lumen by encroachment. Moreover, hypoxia leads to new vessel formation within the pulmonary vasculature and not loss of vessels as formerly thought. Such neovascularization may provide a beneficial adaptation by increasing the area of the gas exchange membrane. These novel structural findings are supported by recent reports that inhibitors of the RhoA pathway can acutely reduce pulmonary vascular resistance in chronically hypoxic lungs to near normal values, demonstrating that structural changes are not the dominant mechanisms underling hypoxic pulmonary hypertension. Chronic hypercapnia inhibits the development of hypoxic pulmonary hypertension, pulmonary vascular remodelling and hypoxia-induced angiogenesis. This last effect might be maladaptive, as it would prevent the potentially beneficial increase in gas exchange membrane area. These findings suggest that structural narrowing of the vascular lumen of resistance vessels is not the mechanism by which hypoxia and hypercapnia cause pulmonary hypertension in chronic lung disease. (Received 22 October 2003; accepted after revision 07 November 2003) Corresponding author P. McLoughlin: Department of Physiology, Conway Institute of Biomolecular and Biomedical Research, University College, Belfield, Dublin 4, Ireland. paul.mcloughlin@ucd.ie Chronic hypoxia caused by migration of native sea level dwellers to high altitude leads to the development of increased pulmonary vascular resistance and pulmonary hypertension (Heath et al. 1973; Rabinovitch et al. 1979; Grover et al. 1983; Meyrick & Reid, 1983; Fishman, 1985; Jones & Reid, 1995). This altitude-induced hypertension offers no obvious benefit and, it has been suggested, may be maladaptive. During the early period of hypoxic exposure, pulmonary vascular resistance is elevated largely due to hypoxic vasoconstriction. However, following sustained exposure to hypoxia, it is thought that structural Presented at a meeting of the Physiological Society at Trinity College Dublin in July changes in the pulmonary vascular bed become the major determinant of elevated vascular resistance (Sime et al. 1971; Lockhart et al. 1976; Fried et al. 1983; Grover et al. 1983; Fishman, 1985). Sustained pulmonary hypertension is a common complication of chronic lung diseases and alveolar hypoxia is thought to be a key stimulus to the development of this complication. Such secondary pulmonary hypertension is strongly associated with increased morbidity and reduced survival (Semmens & Reid, 1974; Ryland & Reid, 1975; MacNee, 1994a,b). Furthermore, evidence of right ventricular hypertrophy in these conditions is an independent predictor of increased mortality, suggesting that pulmonary hypertension contributes DOI: /expphysiol

2 Exp Physiol 89.1pp66 72 Structural basis of pulmonary hypertension 67 directly to increased mortality (Skwarski et al. 1991; Incalzi et al. 1999). In patients with chronic obstructive lung disease (COPD), ventilatory failure leading to chronic hypercapnia in association with hypoxia is a common complication and it is a well-established clinical observation that significant pulmonary hypertension does not develop in the presence of hypoxic lung disease unless hypercapnia is also present (MacNee, 1994a). A close correlation has been noted between the arterial CO 2 tension and the pulmonary arterial pressure (PAP) in this setting (Kilburn et al. 1969; Baum et al. 1971). These observations suggest that elevated P CO2 may contribute importantly to the development of pulmonary hypertension in chronic lung disease although the potential mechanism of this effect is unknown. Here we briefly review the evidence that pulmonary hypertension, secondary to chronic hypoxia, is predominantly caused by structural alterations in the pulmonary circulation. We then consider the view that chronic hypercapnia when it coexists with hypoxia worsens these structural changes thus augmenting pulmonary hypertension. Finally, we present more recent evidence that argues against this paradigm. Altered vascular structure in hypoxic pulmonary hypertension Chronic hypoxic pulmonary hypertension results from sustained vasoconstriction and structural alterations to the pulmonary vascular bed. Following abrupt return to a normal P O2, the immediate fall in pulmonary arterial pressure is small and leaves the arterial pressure elevated substantially above normal values (Sime et al. 1971; Lockhart et al. 1976; Fried et al. 1983). These observations suggested that structural changes in the pulmonary vascular bed are the major determinant of the increased vascular resistance. The structural changes that are thought to underlie the increased vascular resistance can be broadly classified into two processes: (i) remodelling of the pulmonary resistance vessels; and (ii) a reduction in the total number of blood vessels in the lung, sometimes termed rarefaction or pruning. Remodelling results in thickening of the arterial wall and is said to increase resistance by causing the vessel walls to encroach into the lumen and reduce its diameter. This wall remodelling is due to muscularization of previously non-muscular arterioles, increased medial thickness of previously partially and completely muscular arterioles, adventitial hypertrophy and deposition of additional matrix components, including collagen and elastin, in the vascular walls (Rabinovitch et al. 1979; Grover et al. 1983; Fishman, 1985; Stenmark & Mecham, 1997; Rabinovitch, 2001). The second major structural alteration caused by chronic hypoxia is loss of small blood vessels, which is said to increase vascular resistance by reducing the extent of parallel vascular pathways through the lung (Hislop & Reid, 1976, 1977; Rabinovitch et al. 1979; Meyrick et al. 1980; Meyrick & Reid, 1983; Meyrick & Brigham, 1986; Jones & Reid, 1995; Partovian et al. 2000). This loss of blood vessels has been detected as a reduction in the ratio of the number of blood vessels to the number of alveoli in the intra-acinar (gas exchange) regions of the lung. It is interesting to contrast these structural and haemodynamic changes in the pulmonary circulation with those in the systemic circulation in which exposure to chronic hypoxia reduces systemic arterial blood pressure, increases maximal systemic vascular conductance, reduces the vascular response to vasoconstrictors and promotes capillary angiogenesis and arteriogenesis (Hislop & Reid, 1976, 1977; Ruiz & Penaloza, 1977; Rabinovitch et al. 1979; Meyrick et al. 1980; Meyrick & Reid, 1983; Meyrick & Brigham, 1986; Jones & Reid, 1995; Partovian et al. 2000). An alternative view However, not all investigators agree that rarefaction occurs in pulmonary hypertension. Several studies of pulmonary hypertension in the rat report that the ratio of pulmonary arterioles to alveoli was unaltered following chronic hypoxia (Meyrick & Reid, 1979; Emery et al. 1981; Kay et al. 1982; Finlay et al. 1986). More recently, we have re-examined the structural changes induced in the vasculature by chronic hypoxia in the adult rat using the techniques of quantitative stereology combined with confocal microscopy (Howell et al. 2003). Stereology is a technique that allows statistical inferences about the three-dimensional structural parameters of objects based on two-dimensional information such as that provided by histological images (Bolender et al. 1993; Howell et al. 2002). It allows absolute quantities to be measured, for example the total length of intra-acinar resistance vessels in the lung or the total capillary endothelial surface area. It is this ability to make quantitative estimates that distinguishes stereological analysis from conventional histological approaches to assessing changes in vascular structure. In these recent studies, we did not confine our analysis to the pulmonary arterioles alone, a feature common to most previous studies, but also examined pulmonary venules and the gas exchange capillaries within the alveolar walls (Howell et al. 2003). Using these approaches, we found that the total length of

3 68 K. Howell and others Exp Physiol 89.1pp66 72 intra-acinar arterioles and venules together increased rather than diminished. In addition, the mean lumen diameter of these vessels when maximally dilated was not reduced in chronically hypoxic lungs compared to control, although the walls were thickened as previously reported (Howell et al. 2003). Stereological examination of the capillary bed also revealed an increase in total capillary lumen volume, total length, the total endothelial surface area and the total endothelial cell number (Howell et al. 2003). This demonstration of hypoxia-induced angiogenesis in the mature pulmonary circulation implies that we must revise the widely accepted paradigm that hypoxia-induced loss of small vessels is a key structural change contributing to the development of pulmonary hypertension during acclimatization to high altitude environments and in chronic lung disease. Role of hypoxic vascular remodelling in the development of pulmonary hypertension The structural changes in the vasculature that we reported may seem, at first sight, to be incompatible with the observed increase in pulmonary vascular resistance in response to chronic hypoxia. This is not necessarily the case; three mechanisms could reconcile these observations: (i) reduction in lumen narrowing restricted to one critical region of the vasculature; (ii) angiogenesis by elongation; and (iii) differences between vascular dimensions in vivo and in fixed tissues. (i) Our method of lung preparation meant that we could not reliably distinguish between arterial and venous vessels and we therefore considered them together when undertaking stereological measurement. As a result, the mean lumen diameter is a reflection of all the intraacinar vessels excluding capillaries. It is possible that the lumen of the smaller more distal arterioles might have been preferentially reduced in diameter and become a dominant determinant of increased vascular resistance while that of the venous side increased. In this event the mean vessel diameter, as we determined it, could have remained unchanged in hypoxic lungs despite a reduction in the mean diameter of small arterial vessels. (ii) We have shown a marked increase in vessel length in response to hypoxia without significant change in lumen diameter. If this increased length resulted from elongation of existing vessels, as has been shown in other organs (Hansen-Smith et al. 1996; Gambino et al. 2002), and not from formation of new parallel vascular pathways, it would have resulted in a substantial increase in vascular resistance. Thus, the effect of new vessel formation on vascular resistance depends on the exact structural arrangement of these new vessels. New vessels laid down in series would increase vascular resistance whereas new vessels in parallel would reduce it. (iii) The complete distension of vessels prior to fixation produced by our protocol allowed us to investigate the structural changes in these vessels. However, the lumen diameter of the blood vessels determined in this circumstance clearly does not reflect the dimensions that existed in vivo, where in the presence of vascular smooth muscle tone and hypoxic vasoconstriction, the lumen diameter would have been considerably different. Moreover, as in the capillaries of the normal lung, large numbers of these new capillaries may not be perfused at any given moment and thus not have any influence on total resistance. In this context, it is interesting to note the recent report of Nagaoka et al. (2003); these workers found that in chronically hypoxic hypertensive lungs, inhibition of the RhoA/Rho kinase pathway returned pulmonary vascular resistance almost completely to control values. These results suggest that sustained vasoconstriction rather than structural remodelling are the major mechanisms underlying hypoxic pulmonary hypertension. While our findings clearly demonstrate, for the first time, new vessel formation in the pulmonary circulation in response to chronic hypoxia, they are compatible with the development of pulmonary hypertension as a result of vascular remodelling. Nonetheless, pulmonary hypertension does not result from structural loss of blood vessels nor is it likely to result from structural narrowing of the vascular lumen. Effects of hypoxic capillary angiogenesis on gas exchange The changes in capillary structure in the lungs in response to chronic hypoxia may have important beneficial effects on pulmonary gas exchange. Using the model of Weibel et al. (1993) we found that the changes induced by hypoxia caused a significant increase in the total membrane diffusing capacity in chronic hypoxia. In addition, the increase in total capillary volume, by increasing the total volume of blood that could be contained in the lung, might contribute to an increase in total lung diffusing capacity. Finally, the increased capillary length caused by chronic hypoxia, would prolong the time that red blood corpuscles spend in the alveolar capillaries at any given cardiac output, allowing more time for complete equilibration of P O2 between alveolar gas and blood. It must be emphasized, that the inferences made about pulmonary diffusing capacity based on morphometric data give an indication of the theoretical maximum

4 Exp Physiol 89.1pp66 72 Structural basis of pulmonary hypertension 69 diffusing capacity. In vivo these capillaries may not all be perfused and, if perfused, may not be fully distended. Thus, the actual gas exchange surface area and the volume of blood in the lung are unlikely to be as large as those suggested by the morphometric analysis of fixed tissue (Weibel et al. 1993). Furthermore, it must be remembered that when native sea level dwellers first migrate to high altitude, oxygen uptake is not limited by diffusing capacity so that the structural changes in the capillaries would not alleviate resting hypoxaemia. However, in the nonacclimatized sea level native at high altitude, end-capillary P O2 falls substantially below alveolar values leading to further arterial desaturation during exercise (Weibel et al. 1993). Thus, during exercise the increases in capillary dimensions that we have observed would improve oxygen uptake during exercise and, as a consequence, exercise capacity. Effects of chronic hypercapnia As outlined above, chronic hypercapnia is frequently associated with pulmonary arterial hypertension in chronic lung diseases. Yet, in stark contrast with the vast literature on the mechanisms of hypoxia-induced pulmonary hypertension, very few studies of the effects of chronic hypercapnia on the pulmonary circulation have been undertaken, whether acting alone or when combined with hypoxia. Some years ago we compared the effects on the pulmonary circulation in rats of chronic exposure to hypercapnia alone (inspired O 2 fraction (F IO2 ), 0.21; inspired CO 2 fraction (F ICO2 ), 0.10) and hypercapnia combined with hypoxia (F IO2, 0.10, F ICO2, 0.10) to those of hypoxia alone (F IO2, 0.10, F ICO2, 0.00) and controls (F IO2, 0.21, F ICO2, 0.00) using an environmental chamber (Ooi et al. 2000). In rats exposed to hypercapnia alone, there was no increase in pulmonary vascular resistance and no evidence of resistance remodelling. When combined with hypoxia, chronic hypercapnia prevented the development of pulmonary hypertension and vascular remodelling that was observed on exposure to chronic hypoxia alone (Ooi et al. 2000). In view of our recent findings, we were interested to determine the effect of chronic hypercapnia on hypoxiainduced angiogenesis. We exposed male Sprague-Dawley rats from the same colony (Harlan, UK) to either chronic hypercapnia alone or to chronic hypercapnia combined with hypoxia in an environmental chamber, as previously described (Ooi et al. 2000; Howell et al. 2003). Following isolation and fixation of lungs under standard conditions, we used quantitative stereology to assess the resultant changes in pulmonary vascular structure (Howell et al. 2003). In order to facilitate comparison, we reproduce here the previously published results from rats exposed to chronic hypoxia alone and control conditions (Howell et al. 2003). When hypercapnia occurred together with hypoxia, haematocrit, and left and right lung volumes were all greater than in control conditions but significantly less than the values observed in hypoxia alone (Table 1). In addition hypercapnia combined with hypoxia prevented the right ventricular hypertrophy observed as a result of chronic hypoxia acting alone (Table 1). Hypercapnia acting alone did not lead to any significant changes in these values when compared to control conditions. In chronic hypercapnia the mean lumen volume of intra-acinar vessels was not significantly different from that of controls (Table 2). However, mean vessel length was significantly greater in hypercapnia than in the control group suggesting that hypercapnia acting alone leads to angiogenesis, although vessel length was still significantly less than that observed in chronic hypoxia (Table 2). The combined hypoxia and hypercapnia condition did not lead to any further change in total vessel length suggesting that in the presence of hypercapnia, hypoxia was incapable of stimulating new vessel formation (Table 2). Mean vessel length in the hypoxia alone condition was not significantly greater than that in the combined condition (P = 0.07, ANOVA). Neither hypercapnia nor combined hypercapnia and hypoxia caused thickening of the vessel wall in relation to the lumen radius, a finding in good agreement with our previous report of the absence of abnormally remodelled vessels on histological examination in these conditions (Ooi et al. 2000). It is interesting that mean lumen diameter of the maximally dilated vessels, computed as previously described (Howell et al. 2003), was similar in all four conditions examined. This last observation suggests that encroachment of the vascular wall into the lumen does not cause pulmonary hypertension in these conditions. More particularly, chronic hypercapnia does not augment the wall remodelling caused by hypoxia but inhibits this process. The mean capillary volume and mean capillary length per left lung in the hypercapnia alone group were not significantly different from those of control rats (Table 3). In the combined hypercapnia hypoxia group these values were significantly different from those in the hypoxia alone group but not significantly different from those of either the control or hypercapnia alone group (Table 3). As previously reported, chronic hypoxia increased both total alveolar epithelial and total endothelial surface area and the increase in endothelial area was disproportionately greater than the increase in epithelial area (Table 4). When

5 70 K. Howell and others Exp Physiol 89.1pp66 72 Table 1. Body weight, haematocrit, ratio of right to left ventricular plus septum weights and lung volumes in control, hypoxic, hypercapnic and combined hypoxic hypercapnic animals (n = 7) (n = 6) (n = 7) (n = 6) Haematocrit (%) (±0.7) (±1.2) (0.4) (0.5) RV/LV + S Ratio (0.02) (0.03) (0.01) (0.01) Left lung volume (ml) (0.15) (0.24) (0.18) (0.20) Right lung volume (ml) (0.17) (0.21) (0.31) (0.25) Values are mean (± S.E.M.). RV weight, right ventricular weight; LV + S, left ventricular plus septum weight; RV/LV + S, ratio of weight of right ventricular free wall to that of left ventricle plus septum. Significant difference from other three groups (P < 0.01, ANOVA); Significant difference from control group (P < 0.01, ANOVA). Table 2. Lumen volume, vessel length, lumen diameter and ratio of wall thickness to lumen radius of intra-acinar vessels in control and experimental groups (n = 7) (n = 6) (n = 7) (n = 5) Lumen volume (cm 3 left 1 lung) (±0.006) (0.015) (0.007) (0.010) Vessel Length $ (cm left 1 lung) (318) (309) (439) (459) Lumen diameter (µm) (2.4) (4.1) (1.7) (5.7) WT:Radius (0.011) (0.013) (0.005) (0.009) Values are mean (± S.E.M.) WT: Radius, ratio of vessel wall thickness to lumen radius. Significantly different from all other groups (P < 0.05, ANOVA); Significantly different from control group (P < 0.05, ANOVA); $ Significantly different from control and hypercapnic groups (P < 0.05, ANOVA). hypercapnia and hypoxia were present together, these changes were prevented (Table 4); moreover, hypercapnia alone did not lead to any significant changes in these parameters when compared to control values (Table 4). In summary, hypercapnia occurring simultaneously with chronic hypoxia inhibited the capillary angiogenesis observed when chronic hypoxia acted alone. Role of chronic hypercapnia in pulmonary hypertension Our results suggest that hypercapnia inhibits hypoxic vascular wall remodelling in the lungs and should therefore Table 3. Capillary volume and length per left lung in control and experimental groups (n = 7) (n = 6) (n = 7) (n = 6) Capillary volume (cm 3 left 1 lung) (± 0.02) (± 0.03) (± 0.02) (± 0.03) Capillary length ( 10 5 cm 3 per (± 0.60) (± 0.56) (± 0.65) (± 0.81) left lung) Values are mean (± S.E.M.). Control group: F io2, 0.21, F ico2, 0.01, inspired N 2 fraction (F in2 ), 0.79; Hypoxic group: F io2, 0.10; F ico2, 0.01; F in2, 0.90; Hypercapnia: F io2, 0.21; F ico2, 0.10; F in2, 0.69; Combined: F io2, 0.10; F ico2, 0.10; F in2, Significant difference from all other groups (P < 0.05, ANOVA). Table 4. Alveolar epithelial, capillary endothelial surface areas and capillary to alveolar surface area ratio in control, hypoxic, hypercapnic and combined animals (n = 7) (n = 6) (n = 7) (n = 6) Epithelial SA (cm 2 left 1 lung) (± 169) (± 108) (± 113) (± 155) Endothelial SA (cm 2 left 1 lung) (± 186) (± 230) (± 145) (± 235) Ratio of surface areas (± 0.02) (± 0.07) (± 0.02) (± 0.04) Values are mean (± S.E.M.). SA, surface area; ratio of surface areas, ratio of capillary endothelial to epithelial surface area. Significant difference from all other groups (P < 0.05, ANOVA). prevent the development of pulmonary hypertension, an observation that is in keeping with our previous report of the haemodynamic effects of chronic hypercapnia (Ooi et al. 2000). It could be suggested that the development of chronic hypercapnia in COPD is potentially beneficial. However, such actions of hypercapnia seem to be at variance with the positive correlation between arterial P CO2 and pulmonary arterial pressure in patients with COPD and the association of hypercapnia with increased morbidity and mortality. It is important to note that our investigations were concerned with the effect of chronic hypercapnia on hypoxia-induced increases in pulmonary vascular resistance in the absence of coexistent lung disease. In the setting of COPD, other factors may interact with arterial hypercapnia and contribute importantly to elevation of pulmonary arterial pressure. Hypercapnia may act to increase cardiac output thus causing an increase in pulmonary arterial pressure. This effect may be particularly important in COPD as it has

6 Exp Physiol 89.1pp66 72 Structural basis of pulmonary hypertension 71 been demonstrated that, in patients with COPD, resting PAP may be normal or minimally elevated whereas during exercise increased cardiac output leads to abnormally large increases in PAP (MacNee, 1994a). A factor contributing to increased sensitivity to elevated cardiac output in diseased lungs may be reduction in the volume of the pulmonary microcirculation due to parenchymal loss caused by the underlying disease process, for example concomitant emphysematous change and fibrotic obliteration of blood vessels. A second factor augmenting the changes in PAP caused by hypercapnia in patients with COPD may be abnormal airway mechanics. Elevated alveolar pressure augments the change in PAP in response to increased pulmonary blood flow in normal subjects (MacNee, 1994). The abnormal airway mechanics in patients with COPD cause increased alveolar pressure that may interact with high pulmonary blood flows induced by hypercapnia leading to augmented pulmonary hypertension. In the absence of pre-existing parenchymal lung disease, as in the present study, these mechanisms would not act. Direct experimental testing of these possibilities is required. Finally, the greater pulmonary hypertension observed in patients who are both hypoxic and hypercapnic when compared to those who are hypoxic but not hypercapnic may be, in whole or in part, due to more extensive lung damage in such patients and not result from an independent effect of hypercapnia. Effects of chronic hypercapnia on gas exchange We also observed that chronic hypercapnia potently inhibited the induction of capillary angiogenesis by hypoxia (Tables 3 and 4). In the setting of chronic lung disease, this is a potentially maladaptive response. If COPD caused alveolar hypoxia, angiogenesis might improve gas exchange; in the presence of hypercapnia this potentially beneficial action would be lost. Conclusions Although chronic hypoxia causes thickening of the walls of pulmonary arterioles, these changes do not lead to structural narrowing of the lumen by encroachment. Moreover, hypoxia leads to new vessel formation within the pulmonary vasculature and not loss of vessels as formerly thought. Such neovascularization may be a beneficial adaptation by increasing the area of the gas exchange membrane. These novel structural findings are supported by recent reports that inhibitors of the RhoA pathway can acutely reduce pulmonary vascular resistance in chronically hypoxic lungs to near normal values, demonstrating that structural changes are not the dominant mechanisms underling pulmonary hypertension. 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