Hematologic Differences

The Hilltop and Madison strains manifest no hematologic differences under normoxic conditions. However, Hilltop rats develop greater polycythemia in response to hypoxic exposure than Madison rats, with statistically significant differences in hematocrit appearing within two to three weeks (Fig. 1) (23, 24). This is associated with greater increases in total blood volume and red cell mass in Hilltop rats that are apparent within 3 days (Fig. 2A) (35). To compensate for the increase in total blood volume as red cell mass increases, plasma volume decreases similarly in both strains, contributing to hemoconcentration. However, this mechanism is overridden when hematocrit reaches 75%, leading to a rapid increase in total blood volume when hematocrit exceeds that level (35).

Days of Hypoxia

Figure 2. A: Changes in total blood volume (TBV) and red blood cell volume (RBCV) (upper panel) and total plasma volume (TPV) (lower panel) per 100 g body weight (bw) over days of high altitude exposure in Madison (o) and Hilltop (•) rats. *P<0.05 in Hilltop vs Madisons. B: Time course of hematocrit (Hct), mean red cell volume (MCV), reticulocyte count (RETIC) and relative viscosity over duration of hypoxic exposure in days. *P< 0.05 Hilltop vs Madison, fP<0.05 vs. baseline value. SL, seal level (Modified from Refs. 23 and 35).

Days of Hypoxia

Figure 2. A: Changes in total blood volume (TBV) and red blood cell volume (RBCV) (upper panel) and total plasma volume (TPV) (lower panel) per 100 g body weight (bw) over days of high altitude exposure in Madison (o) and Hilltop (•) rats. *P<0.05 in Hilltop vs Madisons. B: Time course of hematocrit (Hct), mean red cell volume (MCV), reticulocyte count (RETIC) and relative viscosity over duration of hypoxic exposure in days. *P< 0.05 Hilltop vs Madison, fP<0.05 vs. baseline value. SL, seal level (Modified from Refs. 23 and 35).

The increased red cell mass parallels an early increase in mean red cell volume in Hilltop rats that is significantly greater than in the Madison strain within 3 days of hypoxic exposure (Fig. 2B). This is accompanied by a greater early reticulocytosis (after 3 days) and a greater increase in relative viscosity of whole blood (after 7 days) in Hilltop compared to Madison rats. The spleen plays a prominent role in extra-medullary hematopoeisis in rats, and splenic size is also greatly enhanced in the Hilltop compared to the Madison rats during exposure to chronic hypoxia (27).

On the other hand, red cell deformability and viscosity of reconstituted blood at equivalent hematocrits did not differ between the strains, suggesting that the Hilltop strain's larger red cells per se do not contribute to the cardiopulmonary differences between the strains. Furthermore, pressure-flow curves of lungs isolated from normoxic and 7 day hypoxic rats suggested that the differences in pulmonary vascular resistance between the strains were related more to pulmonary vascular structural than to hematologic differences (22).

Ou et al. (36, 39) investigated the mechanisms underlying the differences in hypoxia-induced hematological responses between the strains and found increased plasma erythropoietin and renal tissue erythropoietin levels in both strains occurring within 1 day of hypoxic exposure (Fig. 3). However, renal tissue erythropoietin mRNA levels were not different early during the hypoxic exposure. The trend toward greater erythropoietin levels in the Hilltop strain early in the hypoxic exposure reached statistical significance only after 7-10 days of hypoxic exposure. There were no differences in minute ventilation or ventilatory pattern, Pa02, pH, or eryhropoietin clearance from the circulation at any time during the entire 30-day hypoxic exposure . However, the Pa02 and renal venous P02 were lower in the Hilltop compared to the Madison rats from the onset of hypoxia and remained lower throughout.

* m Hintnp

Days of Hypoxia

11.11

Days of Hypoxia

Figure 3. Time course of erythropoeitin plasma levels (A) and renal tissue levels (per gram tissue weight) (B) in Hilltop and Madison rats during days of hypoxic exposure. *P<0.05 Hilltop vs Madison. SL, seal level (Modified from Ref. 36).

The authors performed a post-hoc analysis in which they demonstrated that renal tissue and plasma erythropoietin levels and tissue erythropoietin mRNA levels from both rat strains could be fit on a single dose-response curve in which the erythropoietin response was expressed as a function of renal venous Po2. This implies that the hypoxia sensitivity of erythropoietin synthesis and release in the Hilltop and Madison rats is similar, but Hilltop rats simply experience greater renal venous hypoxia for any level of inspired 02. This leaves unexplained the reason for the greater renal venous hypoxia in the Hilltop rats early during the hypoxic exposure, although it is clearly not related to greater hypoventilation in the Hilltop compared to the Madison rats. Sludging of red cells and slowing of blood flow through capillaries could contribute late during the hypoxic exposure, but this would not explain the early differences, when the strains are similarly polycythemic. In addition, the hypoxia sensitivity of erythropoietin synthesis and release has not been examined explicitly; such a test would require generating the entire dose-response relationship between erythropoietin and renal venous oxygenation - which has not been done. Thus, in addition to the greater renal venous hypoxemia ofthe Hilltop strain, we have not excluded the possibility that erythropoietin synthesis and release is also more sensitive to the hypoxic stress in the Hilltop rats.

To determine whether the enhanced erythropoietic response to hypoxia in the Hilltop rats contributes to its enhanced cardiopulmonary response, Petit et al. (43) administered human recombinant erythropoietin to both strains of rats during hypoxic exposure. They hypothesized that if erythropoietin contributes to the difference, then exogenous administration oferythropoietin to the Madison strain should render its cardiopulmonary responses similar to those ofthe Hilltop strain. Instead, they showed that although hematocrit increased to the same polycythemic level in both strains during hypoxic exposure, the differences in right ventricular peak pressure and right ventricular hypertrophy persisted. In a subsequent experiment, Du et al. (9) demonstrated that hemodilution by phlebotomy to lower mean hematocrit to 46% had no effect on the severity of pulmonary hypertension in the 2 strains. This finding is contrary to what was expected based on the reduction of viscosity, but the authors calculated an increase in vascular hindrance (the vascular resistance to blood viscosity ratio), that was sufficient to counteract the decrease in viscosity. Together, these latter studies indicate that the hematologic differences between the strains that occur during hypoxic exposure do not contribute significantly to the marked hypoxia-induced differences in severity ofpulmonary hypertension and right ventricular hypertertrophy between these strains. By exclusion, these findings suggest that differences in pulmonary vascular responses (i.e., HPV, structural changes or both) are responsible for the cardiopulmonary differences.

2.3. Pulmonary Vascular Differences

2.3.1. Hypoxic Pulmonary Vasoconstriction

Hypoxic pulmonary vasoconstriction (HPV) is characterized by constriction ofpulmonary arterioles in response primarily to alveolar hypoxia that is thought to be a homeostatic mechanism to match perfusion and ventilation for optimizing pulmonary gas exchange. The intensity of HPV differs between and within species and this variation has important effects on adaptation to high altitude. In their original report on the rat strain differences, Ou and Smith (41) observed a striking difference in the severity of pulmonary hypertension between the Madison and Hilltop strains as indicated by their right ventricular peak systolic pressures (RVPP) (74 vs. 50 mmHg in Hilltop vs. Madison, respectively, P<0.05). This difference has been reproduced consistently in subsequent studies on these rats and has been confirmed by direct measurements of mean pulmonary artery pressure (75 vs. 50 mmHg in Hilltop and Madison, respectively, P<0.05) (9). In the earlier study, the peak systolic pressures were probably blunted by anesthesia, offering a likely explanation for why they were essentially the same as the mean pressures measured in unanesthetized animals in the latter study.

Surprisingly, in response to acute hypoxia, the Madison rats have more vigorous HPV, both in isolated lungs (31) and in intact animals (39), in contrast to the greater pulmonary hypertensive responses of the Hilltops to chronic hypoxia. Acute HPV in the Madison strain is very vigorous and is associated with the development of right ventricular aneurysms (39). The occurrence of these lesions is greater with more severe hypoxia, less with mild hypoxia, and reduced by administration of a calcium channel blocker, suggesting that the aneurysms arise from ischemic injury of the right ventricle related to the severity of the acute HPV. These differences in acute hypoxic vasoreactivity between the Madison and Hilltop strains have been replicated not only in isolated blood perfused lungs, but also in isolated pulmonary artery rings (44). In response to severe hypoxia (0% 02 corresponding to a vessel bath Po2 of 10 torr), pulmonary arteries isolated from normoxic Madison rats contracted more that those isolated from Hilltop rats, indicating that the differences in vasoreactivity must be related, at least in part, to differences in the intrinsic properties of the vessels.

Following the acute response, however, pulmonary artery pressures in intact Madison rats follow a biphasic pattern characterized by the initial severe vasoconstriction, followed by a marked blunting of the HPV within 24 hrs during which pulmonary artery pressures return to near normoxic levels (40). This is followed by a gradual rise in pulmonary artery pressure over the next few weeks of hypoxic exposure, but to a much lower level than in the Hilltops. The Hilltop rats have milder acute HPV, but no blunting occurs (40), and pulmonary arterial pressures rise progressively over the next several weeks of hypoxic exposure until severe pulmonary hypertension develops. A "crossover" occurs between 1 and 3 days when the pulmonary arterial pressures in the Hilltop animals exceed those of the Madison rats.

The failure to blunt HPV in the Hilltop rats was confirmed by exposure of intact catheterized animals to acute normoxia. During this test, which serves as an assay of the component of HPV contributing to the elevation of pulmonary arterial pressure, the Madison rats acutely dropped their pulmonary artery pressures by only 2.8 mmHg, consistent with persistent blunting of HPV, whereas the Hilltop rats dropped theirs by 8.6 mmHg (P<0.05 vs. Madison), indicating preservation of HPV (Fig. 4) (40).

The greater acute HPV of the Madison strain that preceeds the greater subsequent blunting is surprising and difficult to explain. The more vigorous acute HPV in the Madison could predispose to more subsequent blunting ofHPV simply by virtue of its magnitude or by some biochemical mechanism, but intuitively, one would hypothesize the contrary; that greater HPV would predispose to greater chronic hypoxic pulmonary hypertension and less HPV to less chronic pulmonary hypertension. This latter view is supported by findings in other hypoxia-adapted species (e.g., pikas and yak) that have blunted HPV and develop only mild altitude-induced pulmonary hypertension (8, 13) and normal altitude-dwelling Tibetans, who exhibit minimal HPV while breathing a hypoxic gas mixture (17). More likely, the ability to blunt HPV is critical in moderating the response to chronic hypoxia in the Madison rats and in other species. As of yet, the mechanism(s) underlying HPV blunting remains unknown, but its identification could be important in understanding physiologic mechanisms of adaptation to hypoxia as well as in giving insights into possible therapies.

Figure 4. Mean pulmonary artery pressure (PAP) values in individual rats at sea level (SL) during acute hypoxic (AH) exposure (10.5% Fio2 for 10 mins) and after 24 hrs (24H) exposure to hypoxia (10.5% F,o2). The Madisons (A) have greater acute hypoxic pressor responses and then blunt these responses more than the Hilltops (B) with sustained hypoxia (Modified from Ref. 40).

Figure 4. Mean pulmonary artery pressure (PAP) values in individual rats at sea level (SL) during acute hypoxic (AH) exposure (10.5% Fio2 for 10 mins) and after 24 hrs (24H) exposure to hypoxia (10.5% F,o2). The Madisons (A) have greater acute hypoxic pressor responses and then blunt these responses more than the Hilltops (B) with sustained hypoxia (Modified from Ref. 40).

2.3.2. Structural Differences

Pulmonary vascular medial thickness differs between species under sea level conditions, and these differences appear to influence subsequent responses to hypoxia. Tucker et al. (47) found a positive correlation between the normoxic medial thickness of small pulmonary arteries and the sensitivity to chronic hypoxia. For example, pulmonary hypertension induced by chronic hypoxia is more severe in cattle and pigs that have greater pulmonary vascular medial thickness, than in dogs, sheep or guinea pigs, that have thinner pulmonary vascular walls (47). Similarly, muscular pulmonary arteries are very thin in animals such as the llama, alpaca, mountain viscacha and Tibetan snow pig that are indigenous to high altitudes and develop only mild hypoxic pulmonary hypertension (51). Further, the native Tibetan yak has successfully acclimated to high altitude by virtually eliminating acute HPV and by having very thin-walled pulmonary vessels (2, 8). The native Himalayan highlanders who have adapted successfully to high altitude also have thin-walled small pulmonary arteries with no medial hypertrophy of the muscular pulmonary arteries or muscularization of the arterioles (18).

The degree of muscularization in response to chronic hypoxia also contributes to the variability in response to chronic hypoxia-induced pulmonary hypertension. The percent increase of medial wall thickness in response to simulated high altitude exposure was significantly less in pika rodents, which did not develop pulmonary hypertension compared to rats that did (13). The Madison and Hilltop rats also exemplify these differences. Although the only difference under normoxic conditions was a greater percentage of partially muscularized alveolar duct vessels in the Hilltop than Madison rats, after 14 days ofhypoxia, the percentage of fully muscular vessels was greater in the Hilltop than Madison rats at both the alveolar wall and alveolar duct levels (31). Furthermore, the percentage increase in medial thickness of preacinar pulmonary arteries in the Hilltop rats was greater than in Madisons. At the intraacinar level, both alveolar wall and respiratory bronchiolar vessels of Hilltop rats had more medial hypertrophy than Madisons. Similarly, Wistar Kyoto rats that develop severe hypoxic pulmonary hypertension have more pronounced vascular remodeling than other strains that develop moderate pulmonary hypertension (1, 49).

These structural differences have functional significance. In pressure-flow experiments in blood-perfused lungs isolated from the Madison and Hilltop strains, Hill et al. (21) demonstrated that pulmonary vascular resistance was greater in hypoxic Hilltop than hypoxic Madison lungs, even after papaverine had been added to the perfusate to eliminate the vasoconstrictive component. This is consistent with the idea that vascular structural differences contribute, along with the differences in vasoconstriction, to the greater pulmonary hypertension in the Hilltop strain. In additional experiments on these strain differences, Colice et al. (6) observed that the greater propensity to pulmonary hypertension in the Hilltop strain is not specific to hypoxia. The plant-derived pyrrole alkaloid, monocrotaline, induced more pulmonary hypertension in the Hilltop than in the Madison strain. Similar to the response to hypoxia, histologic analysis of monocrotaline-injected animals revealed a greater increase in wall thickness of alveolar duct and respiratory brochiole-associated pulmonary vessels in Hilltop than in Madison rats. These differences were not associated with any differences in hematocrit and although Pa02 was slightly less in the Hilltop than Madison rats, the hypoxemia was very mild and insufficient to explain the pulmonary hemodynamic differences. These findings are consistent with the idea that intrinsic properties of the pulmonary vessels rather than exogenous factors are responsible for differing cardiopulmonary responses between strains or species, affecting not only vasoreactivity, but also structural responses.

2.3.3. Cellular Differences

Histologic studies of lungs from chronically hypoxic animals have consistently shown medial thickening suggesting that pulmonary vascular smooth muscle cells proliferate and/or hypertrophy in response to hypoxia, at least in vivo. In cell culture experiments, however, it has been difficult to consistently demonstrate proliferation in response to hypoxic exposure, although some studies have observed potentiation of pulmonary vascular smooth muscle proliferation when hypoxia is combined with other mitogens, such as serum. Furthermore, populations ofpulmonary vascular smooth muscle cells have been identified from the same animal that have differing proliferative responses to mitogens, although these differences have not been attributed to strain differences. In preliminary observations from our laboratory, cultured pulmonary vascular smooth muscle cells from the Madison and Hilltop rat strains proliferated similarly in response to hypoxia and other stimuli, but pulmonary smooth muscle cells from the Madison strain were more responsive to the anti-proliferative effects of atrial natriuretic peptide (Arjona and Hill, unpublished data). This suggests that it is not only responsiveness to mitogenic stimuli, but also the ability to blunt responses to these stimuli that determines strain differences in response to hypoxia, consistent with our in vivo observations.

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Figure 5. Contractile force of intact isolated pulmonary artery rings (A), isolated pulmonary arteries with the endothelium denuded (B), and isolated aortic rings (C) from sea-level Madison and Hilltop rats. All vessel preparations were exposed to 95%, 5%, 3% and 0% 02. *P<0.05 Madison vs Hilltop (Modified from Ref. 40).

Differences in the structure and function of endothelium also appear to contribute to strain variability in the response to hypoxia. Pulmonary artery endothelial cells from the altitude-resistant Yak are much longer, wider, and rounder in appearance compared to domestic cows (hyperresponders to chronic hypoxia) (8). Furthermore, in studies on pulmonary artery rings isolated from the Madison and Hilltop strains, removal of the endothelium abolished the differences in hypoxia-induced contractions (44), suggesting that the strain-related difference in acute HPV is attributable to differences in endothelial function (Fig. 5). This difference was specific for pulmonary vessels; stripping the endothelium from the aortas had no influence on the vasorelaxant responses induced by hypoxia in these vessels. These findings indicate that endothelial cells differ morphologically between species and can contribute to differences in pulmonary vascular reactivity between strains, possibly by means of differences in mediator release.

Figure 5. Contractile force of intact isolated pulmonary artery rings (A), isolated pulmonary arteries with the endothelium denuded (B), and isolated aortic rings (C) from sea-level Madison and Hilltop rats. All vessel preparations were exposed to 95%, 5%, 3% and 0% 02. *P<0.05 Madison vs Hilltop (Modified from Ref. 40).

2.4. Biochemical/Mediator Differences

The differences in pulmonary vasoreactivity and vascular hypertrophy most likely stem from differences in the synthesis, release and/or degradation of vasoactive mediators and mitogens. Likely candidates include the prostaglandins, endothelin, nitric oxide (NO) and other endothelium-derived factors.

2.4.1. Prostaglandins

Prostacyclin (PGI2) is a potent vasodilator prostaglandin synthesized by PGI2 synthase and released from endothelial cells (32). PGI2 also has potent anti-mitogenic effects as well as anti-platelet actions that might be important in moderating pulmonary vascular responses to hypoxia. A relative reduction of release of PGI2 is thought to play a role in the pathogenesis of primary pulmonary hypertension, as such patients have a reduction in the urinary ratio of PGI2 to thromboxane metabolites. Furthermore, Tuder et al. (48) demonstrated that hypoxia-induced pulmonary hypertension is potentiated in mice rendered genetically deficient in PGI2 synthase, indicating that PGI2 is capable of regulating the severity of pulmonary hypertension. However, differences in PGI2 release have not been implicated, as yet, in the mechanism of strain-related differences in cardiopulmonary responses to hypoxia. In isolated pulmonary artery rings from the Madison and Hilltop rat strains, ibuprofen had no effect on the magnitude of acute hypoxic contractile responses (44).

2.4.2. Endothelin-1

Endothelin-1 (ET-1), a potent vasoconstrictor and co-mitogen, plays a significant role in the pathogenesis of hypoxic pulmonary hypertension and pulmonary vascular remodeling. It contributes, at least in some experimental models, to the marked variability between strains in the severity of pulmonary hypertension induced by chronic hypoxia. After hypoxic exposure, ET-1 content in the lung and plasma of hypoxia susceptible strains of rats (Wistar-Kyoto and Fawn-hooded rats) is significantly greater compared to less susceptible strains (Fischer-344 and Sprague Dawley rats) (1, 45). Different ET-1 receptors may also contribute to strain differences. The ET-A receptor mediates vasoconstrictor actions of ET-1, whereas the ET-B receptor mediates both vasoconstrictor and vasodilator (by releasing NO and PGI2) actions. In addition, the ET-B receptor serves a clearance function for ET-1. These properties of the ET-B receptor suggest that its deficiency or blockade could intensify the severity of pulmonary hypertension. Consistent with this idea, rats genetically deficient in the ET-B receptor develop more severe hypoxia-induced pulmonary hypertension than control rats with normal ET-B levels (26).

However, differences in the endothelin system do not explain differences in susceptibility to hypoxia between strains in all instances. Both under normoxic and chronically hypoxic conditions, the Madison and Hilltop rats have similar lung homogenate levels of mRNA and protein content for ET-1 and mRNA content of ET-1 receptors (28) suggesting that other biochemical mediators contribute to the variability in the response to chronic hypoxia.

2.4.3. Nitric oxide

NO, an endothelium-derived vasodilator substance known to be important for the maintenance of low vascular tone in the pulmonary vasculature, is released from l-arginine by the enzymatic action of NO synthase (NOS). Two forms of NOS are found in the lung; endothelial NOS (eNOS), and inducible NOS (iNOS). Reduced expression of eNOS has been reported in lung hemogenates from patients with primary pulmonary hypertension undergoing lung transplant compared to non-pulmonary hypertensive controls (15).

Figure 6. A: Contractile force in pulmonary artery rings isolated from sea-level Madison and Hilltop rats as percent of force generated in response to phenylephrine (PE, 10'6 M). In response to acetylcholine, the Madison rings relax more than the Hilltop rings. *'*'f><0.01 vs. Hilltop. B: Western blots for eNOS and tubulin (upper panels) in lung homogenates from sea-level Madison and Hilltop rats. There was no significant difference in expression between the strains when the eNOS blots were standardized to tubulin (lower panel) (Modified from Refs. 28 and 40).

Figure 6. A: Contractile force in pulmonary artery rings isolated from sea-level Madison and Hilltop rats as percent of force generated in response to phenylephrine (PE, 10'6 M). In response to acetylcholine, the Madison rings relax more than the Hilltop rings. *'*'f><0.01 vs. Hilltop. B: Western blots for eNOS and tubulin (upper panels) in lung homogenates from sea-level Madison and Hilltop rats. There was no significant difference in expression between the strains when the eNOS blots were standardized to tubulin (lower panel) (Modified from Refs. 28 and 40).

Differences in the release of NO could contribute to the differences in hypoxic susceptibility by a number of mechanisms; differences in the levels of substrate (L-arginine), or expression or activity of eNOS or iNOS, or both, although these have not been established as mechanisms for strain differences. In the Hilltop and Madison strains, the greater responsiveness to acetylcholine of pulmonary arteries isolated from normoxic Madison rats suggested that they might be capable of releasing more NO than normoxic Hilltop rats (Fig. 6A). However, pulmonary arteries isolated from the two strains had comparable responsiveness to the direct stimulator of NO release, thapsigargin, and comparable mRNA and protein concentrations of eNOS in lungs of both normoxic and hypoxic animals of the two strains, suggesting that the differing responsiveness to acetylcholine was related to a mechanism not involving the release of NO (28, 29) (Fig. 6B).

2.4.4. Endothelium-derived Hyperpolarizing Factor (EDHF)

EDHF has been described as the component of acetylcholine-induced vascular relaxation that is not blocked by antagonists of NOS, is associated with hyperpolarization, and is blocked by K+ channel antagonists such as apamin and charybdotoxin (EDHF). Evidence for EDHF has been found in a number of vascular beds, including the mesenteric, coronary and pulmonary. In coronary arteries, EDHF has been identified as a product of the cytochrome 450 enzyme, CYP2A. In rabbits, a different cytochrome 450 enzyme, CYP4A, appears to be responsible for the production of EDHF, and 20-hydroxyeicosotetranoic acid has been identified as the likely product. We found evidence for release of more EDHF from pulmonary artery rings isolated from normoxic Madison than from Hilltop rats, based on the greater relaxation of these vessels in response to carbachol, an endothelium-dependent vasodilator, when compared to Hilltop rats (28). The NOS inhibitor, A^'nitro-L-arginine, completely blocked the relaxation response in Hilltop, but not in Madison pulmonary arteries. Furthermore, the residual relaxation in Madison arteries was entirely blocked by apamin and chrybdotoxin, consistent with the hypothesis that the greater relaxation was related to greater release of EDHF. These findings indicate that arteries from the 2 strains have different relaxant responses, possibly related to greater release of EDHF, but this does not explain why the contractile responses to hypoxia are also greater in the Madison strain. Furthermore, although it is tempting to speculate that the greater blunting of pulmonary vasoconstriction during chronic hypoxic exposure in the Madison than Hilltop strain is related to greater release of EDHF in Madisons during chronic hypoxia, we could find no evidence for release of EDHF from isolated pulmonary arteries of either strain after exposure to chronic hypoxia (Hill and Karamsetty, unpublished data).

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