Intermittent Hypoxia Induces Early Functional Cardiovascular Remodeling in Mice

Intermittent Hypoxia Induces Early Functional Cardiovascular Remodeling in Mice Maurice Dematteis 1,2,3 *, Cécile Julien 1,2,4,5 *, Christiane Guillermet 6, Nathalie Sturm 6, Sylvie Lantuejoul 6,7, Michel Mallaret 4, Patrick Lévy 1,2,3 **, and Evelyne Gozal 5,8 ** 1 INSERM ERI17, Grenoble, France; 2 Faculté de Médecine, IFR1, Université Joseph Fourier, Grenoble, France; 3 Laboratoires du Sommeil et EFCR, and 4 Laboratoire de Pharmacologie CEIP, CHU, Hôpital A. Michallon, Grenoble, France; 5 Department of Pediatrics, KCHRI, University of Louisville, Louisville, Kentucky; 6 Département de Pathologie, CHU, Hôpital A. Michallon, Grenoble, France; 7 INSERM U823/UJF, Institut Albert Bonniot, Grenoble, France; and 8 Department of Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky Rationale: Intermittent hypoxia, a hallmark of sleep apnea, is a major factor for hypertension and impaired vasoreactivity. Objectives: To examine the temporal occurrence of these two outcomes in order to provide insight into mechanisms and early cardiovascular disease identification. Methods: Functional and structural cardiovascular alterations were assessed in C57BL6 mice exposed to intermittent hypoxia (21 4% FI O2, 30-s cycle, 8 h/d) or air for up to 35 days. Blood pressure, heart rate, and urinary catecholamines were measured at Days 1 and 14. Hindquarter vasoreactivity was assessed at Days 14 and 35, including vasoconstriction to norepinephrine, endothelium-, and non endothelium-dependent vasodilation. Aorta, heart, and hindquarter skeletal muscles were immunostained for vascular markers PECAM-1 and collagen IV. Measurements and Main Results: Hemodynamic alterations occurred from Day 1, characterized by blood pressure surges with bradytachyarrhythmia driven by cyclic hypoxia. At Day 14, blood pressure at normoxia was elevated, with predominant diastolic increase. With hypoxia, vasopressive catecholamines were elevated, blood pressure surged with a lower hypoxic threshold, whereas heart rate fluctuations decreased. Histologic alterations started from Day 14, with decreased endothelial PECAM-1 expression in descending aorta and left heart. Impaired peripheral vasoreactivity occurred at Day 35, including hypervasoconstriction to norepinephrine secondary to sympathetic hyperactivity, without changes in pre- and postsynaptic a-adrenoceptors or in endothelium- and non endothelium-dependent vasodilation. Conclusions: Intermittent hypoxia induces sequential cardiovascular events suggesting increased chemoreflex and depressed baroreflex, resulting in sympathoadrenal hyperactivity, early hemodynamic alterations with proximal histologic remodeling, and delayed changes in peripheral vasoreactivity. Such early alterations before overt cardiovascular disease strengthen the need for identifying at-risk individuals for systematic treatment. Keywords: sleep apnea; blood pressure; vasoconstriction; histopathology Obstructive sleep apnea (OSA) is characterized by repetitive upper airway occlusions, leading to recurring oxygen desaturations and sleep fragmentation (1). Patients with OSA exhibit increased cardiovascular morbidity and mortality, including (Received in original form February 12, 2007; accepted in final form October 24, 2007) Supported by a grant from Association Grenobloise pour les Insuffisants Respiratoires à Domicile. *These authors contributed equally to this article. **These authors were equally contributing senior authors. Correspondence and requests for reprints should be addressed to Maurice Dematteis, M.D., Ph.D., Laboratoire HP2, Institut Jean Roget, Faculté de Médecine de Grenoble, 38042 Grenoble Cedex 09, France. E-mail: maurice.dematteis@ujf-grenoble.fr This article has an online supplement, which is accessible from this issue s table of contents at www.atsjournals.org Am J Respir Crit Care Med Vol 177. pp 227 235, 2008 Originally Published in Press as DOI: 10.1164/rccm.200702-238OC on October 25, 2007 Internet address: www.atsjournals.org AT A GLANCE COMMENTARY Current Scientific Knowledge on the Subject Intermittent hypoxia in sleep apnea is a major factor for hypertension and impaired vasoreactivity. Understanding the development of these outcomes may condition the management of apneic patients at risk for cardiovascular complications. What This Study Adds to the Field Intermittent hypoxia induced early functional and histologic cardiovascular remodeling in mice. These results strengthen the need for early identification and treatment of apneic individuals at risk for cardiovascular complications. systemic hypertension and ischemic heart and brain diseases (2, 3). OSA is an independent risk factor for hypertension, and several components of OSA may contribute to hypertension, including intermittent hypoxia (IH), hypercapnia, acute negative intrathoracic pressures, and microarousals (3). The role of blood gas alterations has been well characterized (3). Activation of chemoreceptors by either IH or hypercapnia increases sympathetic nervous system (SNS) activity, resulting in blood pressure (BP) and heart rate (HR) surges after each apneic episode (4). Chronic repetition of these acute and short nocturnal episodes leads to increased chemoreceptor sensitivity and SNS outflow and, with the loss of baroreflex compensation (5), increases daytime SNS activity and blood pressure (3, 4). Vascular alterations observed in OSA include impaired endotheliumdependent peripheral vasodilation and increased vasoconstrictor responsiveness (6, 7). Similar to hemodynamic changes, the fact that impaired vasoreactivity can improve with OSA treatment (8, 9) strongly supports a direct relationship between OSA and the vascular abnormalities. However, the temporal relationships between hemodynamic changes and associated vascular alterations associated with OSA are unknown. Although a bidirectional relationship with reciprocal aggravation is conceivable, their respective chronology and contribution to atherogenesis and cardiovascular complications are still unclear. Thus, a better understanding of this pathophysiology may condition the management of apneic patients at risk for cardiovascular complications. Although complete cardiovascular phenotyping of patients with OSA is required to clarify this pathophysiology, clinical investigations may be limited by unknown or confounding factors (disease duration, chronology of events, contribution of the different OSA components, and genetic and environmental factors). Experimental models allow the control of some of these limiting factors and have produced evidence for a causal relationship

228 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 177 2008 between IH and chronic BP increase (10) or vasoreactivity alterations (11, 12). We previously reported an exaggerated vasoconstriction to norepinephrine (NE), whereas the response to acetylcholine was unaffected in mice exposed to 2 weeks of IH (12). The mechanisms underlying these differential alterations, as well as their relationship to hemodynamic changes and cardiovascular tissue remodeling, remain to be elucidated. To address these issues, we studied the chronology and relationships of functional and structural cardiovascular changes in mice exposed to long-term IH up to 35 days. We assessed the acute and chronic changes of BP and HR; we also assessed hindquarter vasoreactivity by investigating the pre- and postsynaptic catecholaminergic transmission for vasoconstriction, and the endothelial and nonendothelial component for vasodilation. Finally, we analyzed histologic cardiovascular alterations at proximal and distal levels. To our knowledge, this is the first comprehensive study evaluating the longitudinal effects of IH on cardiovascular functions and structural remodeling. The findings reported in this study strengthen the need for early identification and treatment of individuals at risk for cardiovascular complications. Some of the results of this study have been previously reported in the form of an abstract (13). METHODS Animals C57BL/6J adult male mice (n 5 158, 8 wk old) were randomized to 1, 14 and 35 days of IH (hypoxic group [HX]: 21 4% FI O2, 30-s cycle, 8 h/d; Days 1, 14, and 35), with respective normoxic control animals (NX group) exposed to air in identical chambers (n 5 8 16 per group). Procedure-related effects were assessed using a second control group of unhandled mice (UN group). Mice were weighed before and on the last day of each week of exposure. Additional details are provided in the online supplement. Hemodynamic Study BP and HR were measured at Days 1 and 14 with a femoral artery catheter, digitized, then analyzed using Acqknowledge software (Biopac Systems, Inc., Santa Barbara, CA). Three periods of recording were selected (Figure 1): (1) a baseline period corresponding to 1 hour air/air exposure, (2) a transition period consisting of the first 15 IH cycles with the hypoxic plateau progressively reaching 4% FI O2, and (3) a 7-hour period of stable IH with all hypoxic plateaus at 4% FI O2. Once the measurements were completed, blood, urine, and tissue were collected for hematocrit, plasma-activated transforming growth factor (TGF)-b1, urine free catecholamines, and immunohistochemistry analysis (see the online supplement). Vascular Reactivity Assessment Vascular reactivity was studied in isoflurane-anesthetized mice at Days 14 and 35 by measuring the perfusion pressure (Pp) from the constantly perfused hindquarter. Vasoconstriction to NE (2, 10, 50 mg) was assessed before and after inhibition of the NE transporter (uptake-1) by desipramine infusion (0.156 mg/min/kg), and after sympathetic inactivation by exsanguination. Vasodilation was assessed in exsanguinated mice with methoxamine-precontracted vasculature (3 mg/kg/min). Acetylcholine (5, 50, 500 ng) was injected before and after blockade of the NO-dependent endothelium vasodilation (L-NAME, 0.2 mg/100 ml for 5 min). Non endothelium-dependent vasodilation was assessed using nitroprusside (200 mg/100 ml) (see the online supplement). Immunohistochemistry Tissues were frozen in liquid nitrogen. Cross-sections (10-mm thickness) of heart, midthoracic aorta, and hindquarter muscle were stained for hematoxylin eosin and sirius red to assess tissue morphology and collagen content. Immunostaining of two vascular markers, platelet endothelial cell adhesion molecule (PECAM)-1 and collagen IV, were performed. Aortic intima-media and adventitia thickness were measured with a Nikon microscope (Eclipse 80i; Nikon France SAS, Champigny sur Marne, France) and LUCIA-G5 software (Laboratory Imaging Ltd, Prague, Czech Republic) (see online supplement). Data Analysis BP was used to determine HR, arithmetic mean BP (MBP), systolic BP (SBP), and diastolic BP (DBP). Pulse pressure was calculated by subtracting DBP from SBP. A double analysis was performed as follows: (1) a global evaluation by averaging the last 15-minute recordings of baseline period and of every hour of IH exposure at Days 1 and 14 and (2) an intracycle analysis of both transition and stable IH periods by comparing the hemodynamic fluctuations during normoxic and hypoxic plateaus (NxP and HxP, respectively). For the transition period, because the HxP progressively decreased to 4% FI O2, hemodynamic response was assessed for each of the 15 cycles. For the stable IH period, because the HxP were all at 4% FI O2, values from NxP or HxP of the last 10 cycles of each hour were averaged. Results were expressed as mean 6 SEM and analyzed using Wilcoxon and Mann-Whitney tests for intragroup and intergroup comparisons, respectively; a P value less than 0.05 was considered significant. RESULTS Body Weight Evolution Initial body weights were similar in both mouse groups (Figure 2). NX mice progressively gained weight. In contrast, weights of HX mice initially decreased then increased parallel to NX mice but remaining significantly lower. Figure 1. Schematic representation of the three periods of hemodynamic measurements. BP 5 blood pressure; HR 5 heart rate; IH 5 intermittent hypoxia.

Dematteis, Julien, Guillermet, et al.: Hypoxia and Cardiovascular Changes 229 Figure 2. Body weight evolution during intermittent hypoxia (IH). Body weight alteration in mice exposed to 35 days of IH (HX) or air/air (NX), n 5 16 per group; P, 0.05 for intergroup* and intragroup comparisons. 140.1 6 4.46 vs. 126.2 6 5.45 mm Hg). No significant difference in BP and HR emerged between NX and UN mice at Day 14. Hemodynamic measurements during the transition period. During this period, BP and HR were measured at NxP and HxP of each IH cycle (C1 to C15) to assess cardiovascular responses to progressive FI O2 decline, during acute (Day 1) and midterm (Day 14) IH exposure (Figure 4). This allowed to assess chemoresponses for different levels of oxygen desaturation. At Day 1, BP increased (from 111.8 6 4.95 to 119.3 6 7.27 mm Hg) when FI O2 reached 8.5% and remained elevated for three cycles, even during NxP. BP then progressively decreased, with oscillations driven by cyclic hypoxia from 6.7% FI O2 corresponding to BP surges at the end of the hypoxic plateaus (Figure 4B). At Day 14, baseline BP was elevated and further increased only during hypoxic phases. These hypoxic BP surges started from 12.2% versus 8.5% FI O2 at Day 1, with maximum oscillations and elevations at 8.5%. BP subsequently decreased, but more slowly than at Day 1, thus remaining elevated at the end of the transition period. BP and HR Hemodynamic changes were assessed during three different periods reflecting daytime period, sleep onset, and nighttime sleep in patients with OSA, as shown in Figure 1. Hemodynamic measurements at baseline. NX and HX animals had similar MBP and HR at Day 1, whereas at Day 14, HX mice had increased MBP with a trend for decreased HR (P 5 0.06) compared with NX and UN mice (Figure 3). DBP increased more (115.1 mm Hg; HX vs. NX, 122.3 6 4.12 vs. 107.1 6 5.82 mm Hg) than SBP (113.9 mm Hg; HX vs. NX, Figure 3. Hemodynamic parameters at baseline. Mean arterial blood pressure and heart rate at baseline period, before any exposure to intermittent hypoxia (Day 1) and at Day 14, in hypoxic (HX; n 5 11 at Day 1, n 5 10 at Day 14), normoxic sham (NX; n 5 9 at Days 1 and 14), and unhandled (UN; n 5 8) mice. *P, 0.05 versus NX. Figure 4. Hemodynamic parameters during the transition period. (A) FI O2 recording from an individual chamber. (B) Mean arterial blood pressure at Day 1 (D1; circles) and Day 14 (D14; squares) of intermittent hypoxia (IH). (C) Heart rate (HR) at Days 1 (circles) and 14 (squares) of IH. Dotted lines represent minimum and maximum HR at Day 1 and Day 14. Note the larger HR fluctuations at Day 14 compared with Day 1(long vs. short double-headed arrows between the dotted lines). For both B and C, normoxic and hypoxic plateaus were compared; *P, 0.05. Ten and 11 hypoxic mice were used at Days 1 and 14, respectively. HxP 5 hypoxic plateau; NxP 5 normoxic plateau.

230 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 177 2008 HR fluctuations were minimal at Days 1 and 14 until reaching 8.5% FI O2, then increased with greater amplitude at Day 14 versus Day 1, independently from the normoxic or hypoxic phases (Figure 4C). Hemodynamic measurements during 7 consecutive hours of IH. At Day 1, BP progressively decreased throughout the 7 hours in both HX and NX mice (Figure 5A). Motor activity also decreased over the first 30 minutes of IH, after which the animals remained mainly asleep. This behavioral pattern persisted at Day 14. In contrast to Day 1, Day 14 HX mice had higher baseline BP with a steeper decrease throughout the 7 hours of assessment (Figures 5A and 5B). BP elevation in HX mice was predominantly diastolic, and decreased more rapidly in HX mice, as substantiated by slope calculations (HX vs. NX: SBP, 21.71 6 0.78 vs. 20.88 6 0.54 mm Hg/h; DBP, 22.07 6 0.92 vs. 21.45 6 0.47 mm Hg/h) (Figures 5C and 5D). Pulse pressure was lower in HX mice at Day 14 (HX vs. NX: 15.2 6 2.90 vs. 22.6 6 2.34 mm Hg; P 5 0.072), and progressively increased throughout the 7 hours in both groups to reach 19.5 6 4.60 and 25.9 6 2.31 mm Hg for HX and NX mice, respectively. In contrast, no difference in HR emerged between HX and NX. To assess baroresponses, we measured hemodynamic fluctuations during NxP and HxP at each hour of the 7-hour recording period. We observed similar amplitude of hypoxic BP surges at Days 1 and 14 (Figures 5E and 5F). However, BP oscillations at Day 14 occurred with reduced HR variability compared with Day 1. Figure 5. Hemodynamic parameters during stable intermittent hypoxia (IH). Mean arterial blood pressure (BP) at Days 1 (A) and 14 (B) in hypoxic (HX) and normoxic (NX) mice. Systolic (C) and diastolic (D) BP at Day 14 in HX and NX mice. HX versus NX mice, *P, 0.05. BP and heart rate (HR) fluctuations during hypoxic versus normoxic plateaus at Days 1 (E) and 14 (F). Data are expressed in percentage of normoxic plateau value for each hour of the 7 hours of measurement, and statistical analysis was performed on raw data from hypoxic versus normoxic plateaus; *P, 0.05. Ten HX and eight NX mice were used at Day 1; 11 HX and 6 NX mice were used at Day 14. B 5 baseline period; T 5 transition period.

Dematteis, Julien, Guillermet, et al.: Hypoxia and Cardiovascular Changes 231 Biochemistry Hematocrit, TGF-b1, and catecholamines were determined at the end of the 8-hour exposure at Days 1 and 14 (Table 1). At Day 14, HX animals had higher hematocrit than NX mice. No significant difference in TGF-b1 appeared between HX and NX animals at Days 1 and 14. Free dopamine and NE were higher in some HX mice at Day 1, whereas epinephrine was elevated in some HX mice at Day 14. This was confirmed by the catecholamine ratios, reflecting a delayed increase in epinephrine versus NE, and prominence of the two vasopressive catecholamines over the dopaminergic system at Day 14. Vascular Reactivity Vasoconstriction. NE induced similar dose-dependent responses in Day 14 HX and Day 14 NX mice. In contrast, Day 35 HX mice had higher baseline Pp (P 5 0.07) and enhanced NE response compared with NX mice (Figure 6A and Figure E1 of the online supplement). This increased responsiveness was significant from 10 mg NE and reached a plateau for the two highest doses. Contribution of NE clearance to this exaggerated response was assessed with the NE reuptake inhibitor desipramine (Figure 6B and Figure E1). Desipramine and exsanguination similarly lowered baseline Pp and the vasoconstriction response to NE at all doses, without a significant difference between NX and HX groups at Days 14 and 35 (Figures 6B and 6C, and Figure E1). Vehicle injection led to similar Pp than baseline values, and no difference between NX and UN animals emerged. Vasodilation. During methoxamine precontraction, baseline Pp and response to acetylcholine was similar in HX mice and in their respective control group (Figures 6D and 6E, and Figure E2). NO synthesis inhibition by L-NAME produced similar elevation in baseline Pp and response to acetylcholine in HX and NX mice, suggesting no alterations in endothelium-dependent vasodilation (Figure 6E and Figure E2). Assessment of endothelium-independent vasodilation with nitroprusside did not reveal any Pp difference between HX and NX animals (Figure 6E and Figure E2). Vehicle injection led to similar Pp than baseline values regardless of the group and time point. Histologic Cardiovascular Remodeling HX mice exposed to 14 days of IH exhibited decreased PECAM-1 immunostaining at the aortic and cardiac levels, whereas no difference emerged for hindquarter skeletal muscles at 14 and 35 days of IH. At the aortic level, the decreased endothelial PECAM-1 expression predominated at the dorsal wall of intima, without evidence of endothelial denudation (Figure 7A). Similar reduction in staining occurred in the left ventricular myocardium, with a gradient from the pericardium to the endocardium, and almost no staining in the deepest layers of the ventricle of some animals, especially in the papillary muscles (Figure 7B). Comparatively, the right ventricle was less affected or not affected. Collagen IV staining showed no difference in heart collagen density between HX and NX animals at Day 14, suggesting no difference in capillary density. Regarding intima-media thickness, although HX mice tended to have higher values at Day 14, implicating the media, no significant difference emerged, as for adventitia thickness (Figure 7C). DISCUSSION To clarify the pathophysiology of OSA-related cardiovascular complications, we studied the chronology of functional and structural cardiovascular changes and their relationship in mice exposed to long-term IH. Hemodynamic alterations occurred first at Day 1, as BP and rhythmic oscillations driven by IH cycles. After 2 weeks of IH, BP was elevated at normoxia with predominant diastolic increase. With hypoxia, sympathoadrenal activity was enhanced; BP surged with lower oxygen desaturation, whereas HR fluctuations decreased. Proximal histologic cardiovascular remodeling appeared at Day 14, with decreased endothelial PECAM-1 expression in the descending aorta and left heart. Distal functional vascular remodeling became obvious at Day 35, characterized by increased vasoconstriction to NE, with evidence of sympathetic hyperactivity without changes in pre- and postsynaptic a-adrenoceptor vasoresponse, or in endothelium- and non endothelium-dependent vasodilation. Methodologic Considerations Considering their high breathing frequency and metabolic rate, we exposed mice to 30-second IH cycles (i.e., 120/h of apnea index), which compares with severe human OSA. Moreover, this short cycle duration limited the hypoxia-induced hyperventilation that may induce hypocapnia and cardiovascular changes. We selected a 4% FI O2 nadir based on our experience and previous studies showing that FI O2 of less than 5% produced maximal changes in BP and HR (14). We did not record sleep to show sleep disruption, which may have affected our results. We exposed mice to IH during daytime, which is the preferential sleep period in rodents. On the basis of behavioral assessment, TABLE 1. BIOCHEMICAL ALTERATIONS INDUCED BY INTERMITTENT HYPOXIA Day 1 Day 14 Hypoxic Mice Normoxic Mice Hypoxic Mice Normoxic Mice Blood Hematocrit, % 41.3 6 1.90 (n 5 8) 41.0 6 2.38 (n 5 6) 46.3 6 1.20 (n 5 10)* 38.7 6 1.29 (n 5 10) TGF-bl, ng/ml 6.88 6 0.91 (n 5 4) 5.43 6 0.67 (n 5 5) 4.98 6 0.34 (n 5 10) 5.27 6 0.76 (n 5 12) Urine Epinephrine, nmol/l 40.3 6 15.2 (n 5 7) 34.2 6 13.2 (n 5 6) 549 6 253 (n 5 7) 164 6 93.9 (n 5 9) Norepinephrine, nmol/l 154 6 46.2 (n 5 7) 104 6 29.7 (n 5 6) 269 6 105 (n 5 7) 145 6 53.5 (n 5 9) Dopamine, nmol/l 1,063 6 351 (n 5 7) 341 6 207 (n 5 6) 684 6 246 (n 5 7) 898 6 297 (n 5 9) E/NE ratio 0.68 6 0.22 (n 5 7) 0.42 6 0.12 (n 5 6) 3.20 6 1.06 (n 5 7)* 0.77 6 0.29 (n 5 8) (NE 1 E)/DA ratio 0.33 6 0.14 (n 5 7) 0.66 6 0.15 (n 5 6) 1.35 6 0.15 (n 5 7)* 0.51 6 0.10 (n 5 8) Definition of abbreviations: DA 5 dopamine; E 5 epinephrine; NE 5 norepinephrine; TGF-b1 5 transforming growth factor-b1. Hematocrit, TGF-b1, and urinary catecholamines measured after 8 hours of intermittent hypoxia (hypoxic mice) or air/air (normoxic mice) exposure at Days 1 and 14. * Comparison between hypoxic and normoxic mice, P, 0.05. Comparison between hypoxic mice at Days 1 and 14, P, 0.05.

232 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 177 2008 Figure 6. Vasoreactivity after intermittent hypoxia (IH). Vasoconstriction (A D). Vascular responses to norepinephrine (NE) in mice exposed to 14 and 35 days of IH (HX14, n 5 8; HX35, n 5 7) or air (NX14, n 5 7; NX35, n 5 8). (A) Before NE-reuptake blockade and hemorrhage-induced sympathetic inhibition; (B) with desipramine inhibition of NE reuptake; (C) with desipramine NE-reuptake blockade and sympathetic inactivation by exsanguination; (D) vascular response to methoxamine. *P, 0.05. See also Figure E1 of the online supplement. Vasodilation (E) in mice exposed to 14 and 35 days of IH (HX14, n 5 8; HX35, n 5 7) or air (NX14, n 5 7; NX35, n 5 7). Mice were exsanguinated to inhibit sympathetic activity. Vascular response to acetylcholine (Ach; 500 ng) with active nitric oxide (NO) synthesis (left), and with inactive NO synthesis (L-NAME L-nitro-arginine methyl ester) (middle). Note that three doses of Ach were tested (5, 50, 500 ng), but only the highest dose is shown in the figure. Non endotheliumdependent vasodilation with the NO donor sodium nitroprusside (200 mg) (right). See also Figure E2. HX14 5 Day 14 hypoxia; HX35 5 Day 35 hypoxia; NX14 5 Day 14 normoxia; NX35 5 Day 35 normoxia. our mice spent most of the time sleeping during IH exposure, although microarousals could not be ruled out. However, rodents have polyphasic sleep; they do not sleep for 8 consecutive hours. They also sleep during nighttime, and are able to compensate for daytime sleep disruption by shifting their preferential sleep period to nighttime. Overall, this suggests that our results were mainly due to hypoxia. Hemodynamic Changes Baseline period. The baseline period is representative of the daytime period in patients with OSA. After 2 weeks of IH, mice had elevated BP during normoxia. This finding provides new evidence for a causal relationship between IH and the rapid development of systemic hypertension independently of confounding factors. The magnitude of BP increase, with its predominant diastolic component, is in agreement with human OSA pathology (2, 3, 15). The pathophysiology may involve recurrent sympathetic stimulations through carotid and aortic chemoreceptor activation at each oxygen desaturation, leading to chronic SNS hyperactivity and increased circulating catecholamines (3, 4). Urinary catecholamines, reflecting integrative sympathoadrenergic activity during IH exposure, uncovered a delayed increase in epinephrine. Although NE preferentially reflects SNS activation, epinephrine mainly characterizes adrenal involvement (16), suggesting differential regulation of these two components in response to acute and chronic hypoxia (17). The vasoreactivity study suggested SNS hyperactivity (see below). Together, these changes should result in increased BP, whereas HR should decrease due to vagally mediated inhibition of baroreceptors. However, we found evidence of reduced baroreflex (see below). Alternatively, the decreased HR observed in this study may result from changes in blood volume and redistribution. Venous blood return can be increased in OSA, due to peripheral vasoconstriction and augmented renal sodium reabsorption secondary to renin angiotensin system activation and negative intrathoracic pressure (3). These changes could lead to intermittent change in ventricular pre- and afterload, diastolic dysfunction, and enhanced DBP (3). Transition period. The transition period represented the apneic episodes at sleep onset and allowed assessment of cardiovascular responses during progressive oxygen desaturation. After 2 weeks of IH, BP surges occurred at a less severe hypoxia, with slower return to baseline, and greater BP and HR instability than at Day 1. Such alterations may underlie arrhythmic complications associated with OSA (15) and may involve chemoreceptor modifications. Carotid bodies are the major peripheral sensors for detecting oxygen decline in arterial blood, and chronic IH may lead to increased carotid body sensitivity and response to hypoxia through long-term facilitation (18). Stable IH period. Representative of the human nighttime period, the stable IH period was characterized by a progressive decline in BP over the 7 hours in all groups; however, this was more pronounced in the chronic IH animals. Such daytime fall in BP is typical in rodents and may relate to their reduced motor activity during sleep (19). In addition, sleep regulation itself may contribute to this decreased BP through changes in the autonomic balance (increased parasympathetic vs. decreased sympa-

Dematteis, Julien, Guillermet, et al.: Hypoxia and Cardiovascular Changes 233 Figure 7. Histologic cardiovascular alterations induced by 14 days of intermittent hypoxia. (A) Platelet endothelial cell adhesion molecule (PECAM)-1 immunostaining of aorta cross-sections (10 3 10 magnification; insets, 103 40 magnification) decreased in hypoxic (HX) versus normoxic (NX) mice, without endothelial denudation as suggested by the presence of endothelial cell nuclei (10 3 40 magnification, arrows). (B) PECAM-1 immunostaining of heart cross-sections (10 3 10 magnification) decreased in HX versus NX mice, with a gradient from the periphery to the center of the section (dotted triangle). Collagen IV and PECAM-1 immunostainings of heart cross-sections are shown in 10 3 40 magnification insets; only PECAM-1 expression decreased in HX mice (C) Sirius red staining of aorta cross-sections. Left inset shows measurements of the wall thickness (double-headed arrows). Right inset shows that intima media thickness in HX mice was not significantly different from NX mice. Four to five mice per group were used for immunostainings. thetic activity) (3). However, despite their greater BP decrease, BP was still elevated in Day 14 HX mice compared with Day 14 NX mice. Thus, similarly to apneic patients, hypoxic mice suffered from enhanced BP throughout the nycthemeral cycle. In addition, the sleep-related BP fall can disappear in patients with OSA (15, 20). This nondipping pattern seems to be highly predictive of cardiovascular complications even without daytime hypertension (3). In contrast, Day 14 HX mice exhibited a greater BP decrease. Beyond a species-related difference, one explanation may rely on dopamine. Urinary free dopamine stems from a local renal dopaminergic natriuretic system, derived from circulating dopa and acting as an autocrine/paracrine substance (16) that may participate in the hypoxic diuretic response (21). HX mice at Day 1 had the highest dopamine levels and the lowest (norepinephrine 1 epinephrine)/dopamine ratio, illustrating a predominant secretion of dopamine. In contrast, Day 14 HX mice were not different from NX mice for dopamine, and their catecholamine ratio was inverted compared with Day 1, reflecting the predominant secretion of the two vasopressive catecholamines. Alternatively, hypoxic vasodilation, involving the adenosine prostaglandin NO cascade (22), may account for the greater DBP decline. Indeed, adenosine accumulates during hypoxia resulting from ATP degradation. However, such compensatory vasodilation is transient, gradually decreasing over days, and is replaced with neoangiogenesis to alleviate tissue hypoxia. In that case, the nondipping pattern could occur at a later stage of IH exposure. Assessment of the hemodynamic variability throughout 7 hours showed similar BP fluctuations after acute and chronic IH, but with reduced HR variability in chronic IH mice. Although the hemodynamic alterations concomitant with the FI O2 decline of the transition period may relate to increased chemosensitivity, BP oscillations with reduced HR fluctuations suggest depressed baroreflex, due to either decreased sensitivity or increased set point (3, 5). Functional Vascular Remodeling The enhanced vasoconstrictor response to NE with normal endothelium- and non endothelium-dependent vasodilation agrees with previous studies (7, 12), although others have demonstrated impaired vasodilation (8, 9, 11). The discrepancies may originate from our experimental procedure. We assessed in situ vascular response of the entire vessel network with its surrounding regulation. This setting, as well as the effect of isoflurane anesthesia, may have masked the endothelial dysfunction previously detected in isolated artery (11). Furthermore, the characteristics of the hypoxic stimulus may also affect the result. In our previous study, the same IH paradigm in a noisier apparatus led to a similar but earlier increased vasoconstriction to NE (12), suggesting that IH with an additional stress factor may be more detrimental. In contrast, different hypoxic stimuli may induce an opposite response, as chronic sustained hypoxia in rats has led to decreased vasoconstrictor responses (23). The delayed changes in vasoreactivity suggest a phenomenon secondary to

234 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 177 2008 both hypoxia and hemodynamic alterations. The exaggerated response to NE may be due to the following: (1) an increased NE release and or (2) a decreased NE clearance and/or (3) postsynaptic alterations. Increased NE release, a corollary of SNS hyperactivity/ hyperreactivity, is well established in OSA (3, 4) and agrees with our results suggesting increased chemoreflex. Higher catecholamine levels may result from increased release and/or decreased clearance. NE reuptake via the NE transporter uptake-1 is the main mechanism for NE clearance (16). Such an energy-dependent mechanism can be affected by hypoxia and oxidative stress. We used desipramine to inhibit the NE reuptake leading to NE accumulation in the synaptic cleft. This resulted in presynaptic a 2 - receptor mediated inhibition of SNS activity with decreased NE release and subsequent decreased baseline Pp. Desipramine induced similar decreased baseline Pp in HX and NX mice and lowered pressor responses to NE at all concentrations without differences between groups. This suggests that NE-reuptake inhibition was similar between groups and that presynaptic a 2 - receptors were likely not involved in the differential NE response of Day 35 HX mice. Exsanguination did not further enhance sympathetic inhibition, suggesting that the desipramine sympathoinhibitory effect was maximum. Postsynaptic alterations may result from altered number and/or affinity of postsynaptic a-adrenoceptors. However, HX and NX mice exhibited similar responses to NE after SNS suppression, or after administration of the a 1 -agonist methoxamine. Overall, the hyperresponsiveness to NE appeared to result from SNS hyperactivity. Histologic Cardiovascular Remodeling Proximal cardiovascular remodeling occurred from Day 14. We used two vascular markers, PECAM-1 for endothelial cells and collagen IV for perivascular basement membranes, but only PECAM-1 was modified. Affected areas included the descending aorta with no evidence of endothelium denudation, and the left ventricle, predominating in the deepest layers without decreased capillary density, whereas the right ventricle was less affected. This topographic distribution pattern of decreased PECAM-1 expression may result from hemodynamic strains induced by IH. PECAM-1 is a cell cell adhesion molecule most abundantly expressed in endothelial cells and previously believed to mediate cell adhesion, neogenesis, and leukocyte transmigration through the endothelium (24). Recent evidence implicates PECAM-1 as a mechanoreceptive molecule (25). Indeed, shear stress triggers a rapid tyrosine phosphorylation of PECAM-1, initiating a signaling cascade leading to enos activation (26). Chronic exposure to biomechanical forces may alter PECAM-1 expression and distribution, which in turn may represent an important mechanism modulating endothelial cell sensitivity to mechanical stimuli (25). Shear stress is indeed a critical determinant of cardiovascular homeostasis, regulating remodeling and atherogenesis. Although the vasoreactivity experiment showed no evidence of peripheral endothelial dysfunction, PECAM-1 alterations implicate the aortic and cardiac endothelium in IH pathophysiology. Whether down-regulated endothelial PECAM-1 expression in our HX mice represents a marker of endothelial dysfunction or an early event of atherogenesis (27, 28) remains to be determined. Large-artery stiffness increases pulse pressure, which is associated with several cardiovascular risk factors (29). However, we observed a decreased pulse pressure resulting from the prominent DBP increase. Increase in pulse pressure may occur later, following appearance of structural remodeling leading to arterial stiffness. We assessed TGF-b1 as an early marker of tissue remodeling. TGF-b1 is a potent antiinflammatory cytokine involved in angiotensin-mediated tissue remodeling, promoting collagen expression and fibrosis (30). In agreement with clinical studies (31), no difference in TGF-b1 emerged between HX and NX mice. However, normal plasma TGF-b1 cannot exclude localized tissue TGF-b1 alterations. Cardiovascular Alterations Induced by IH: Pathophysiologic Hypothesis Collectively, our results suggest the following pathophysiology that may occur in OSA. IH activation of chemoreceptors leads to increased SNS tone resulting in BP surges with subsequent baroreflex-mediated bradyarrhythmia. Proximal cardiovascular remodeling as evidenced by decreased PECAM-1 staining occurs early, whereas peripheral functional vascular remodeling as shown by the increased vasoconstriction to NE is delayed. While the proximal PECAM-1 changes suggest a prominent effect of hemodynamic strains, the respective contribution of IH and hemodynamic alterations to the delayed hypervasoconstriction remains to be determined. These alterations, in turn, may contribute to persistent hemodynamic changes. In addition, increased BP becomes permanent because of enhanced chemoreceptor sensitivity and sympathoadrenal activity, whereas baroreceptor response is decreased. Peripheral vascular remodeling may further become structural, eventually leading to atherogenesis, and resulting in chronic tissue hypoxia; together with impaired coagulation and augmented hematocrit levels, this may increase risks for acute ischemic and arrhythmic complications (3). 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