Potential conflict of interest: Nothing to report.
Supported in part by grants from Fondos FEDER. M. H.-G. is the recipient of a Grant from Instituto de Salud Carlos III (538/07) and Programa de Intensificación de Actividad Investigadora (INT07/173).
Chronic intermittent hypoxia (CIH) occurs with obstructive sleep apnea syndrome (OSAS) and provokes systemic endothelial dysfunction, which is associated with oxidative stress and low nitric oxide (NO) bioavailability. Cirrhotic livers exhibit intrahepatic endothelial dysfunction, which is characterized by an impaired endothelium-dependent response to vasodilators and hyperresponse to vasoconstrictors. We hypothesized that CIH may also contribute to intrahepatic endothelial dysfunction in cirrhosis. Normal and cirrhotic rats were exposed for 14 days to repetitive cycles of CIH mimicking OSAS in humans, or caged with room air (handled controls [HC]). Hepatic endothelial function was assessed in isolated and perfused rat livers by dose-response curves to acetylcholine (ACh) and methoxamine (Mtx). In a group of cirrhotic rats, in vivo systemic and hepatic hemodynamic parameters were evaluated at baseline and after volume expansion. In addition, liver samples were obtained to assess endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS), NO bioavailability, and nitrotyrosinated proteins as a marker of oxidative stress. Cirrhotic rats exposed to CIH exhibited an attenuated vasodilatory response to ACh and hyperresponse to Mtx compared with HC rats. During volume expansion, similar portal pressure increases were observed in CIH and HC rats, although the mean arterial pressure increase was lower after CIH. These functional responses were associated with the presence of increased hepatic oxidative stress without changes in p-eNOS after CIH exposure. In normal rats, no hemodynamic changes were found. Conclusion: CIH exacerbates intrahepatic endothelial dysfunction in cirrhotic rats, which is associated with increased oxidative stress that may reduce NO bioavailability. Clinical studies are needed to assess whether OSAS contributes to endothelial impairment in human patients with cirrhosis. (HEPATOLOGY 2013;57:1564–1574)
Intrahepatic endothelial dysfunction is regarded as a key early event in liver cirrhosis. This impairment is characterized by an abnormal nitric oxide (NO) endothelium-dependent relaxation and an exaggerated response to vasoconstrictors in the hepatic vascular bed. Both factors contribute to increase hepatic vascular resistance, leading to portal hypertension and its complications.1, 2
Obstructive sleep apnea syndrome (OSAS) is characterized by chronic intermittent hypoxia (CIH) and also provokes systemic endothelial dysfunction, as suggested by reduced endothelium-dependent vasodilation. Indeed, several clinical studies have demonstrated reduced flow-mediated dilation3 and blunted vasodilation in response to acetylcholine (ACh),4 which acts on the endothelium and causes vasodilation through an NO-dependent pathway. In addition, experimental studies using animal models of CIH have shown attenuation in the vascular response to ACh in different vessels5 and increased vasoconstriction.6 These responses have been attributed to increased scavenging of NO by reactive oxygen species (ROS), because CIH is characterized by a repetitive decrease in tissue oxygen supply and reoxigenation that generates oxidative stress.7
Reduced NO availability in this setting, and the resulting endothelial dysfunction, underlie OSAS-related cardiovascular risk. In fact, CIH and OSAS have been identified as independent risk factors for cardiovascular diseases such as systemic arterial hypertension, myocardial infarction, and stroke.8 In addition, OSAS is frequently associated with metabolic syndrome (obesity, insulin resistance, and hypertension), which itself may aggravate the endothelium impairment.9
CIH has also been shown to occur in patients with cirrhosis due to the presence of ascites10, 11 and obesity.12 More recently, CIH has been shown to be highly prevalent among patients with hepatopulmonary syndrome, and it has been associated with poor prognosis.13
It is therefore possible that oxidative stress produced by CIH decreases NO bioavailability and results in attenuation in vasodilation and hyperresponse to vasoconstrictors, contributing to the observed increase in hepatic vascular resistance of cirrhotic livers. Thus, the present study aimed to investigate the role of CIH in modulating hepatic vascular tone in normal and cirrhotic rats, focusing on two animal models of cirrhosis at different disease stages, and the possible mechanisms involved.
Materials and Methods
Age-matched male Sprague-Dawley rats weighing 175-300 g before beginning the exposure to intermittent hypoxia or air-air cycling were used.
We used carbon tetrachloride (CCl4) and common bile duct ligation (CBDL) models to evaluate the role of CIH in two different models of cirrhosis.
CCl4 Cirrhotic Animals.
A group of rats weighing 175-300 g underwent inhalation exposure to CCl4 pretreated with phenobarbital (0.3 g/L) to accelerate fibrosis for a period of 8 and 12 weeks (early and advanced cirrhosis, respectively).14 CCl4 inhalation was then interrupted and the animals were randomly allocated in cages for CIH exposure protocol.
CBDL Cirrhotic Animals.
Rats weighing 230-280 g underwent bile duct ligation as described15 (see Supporting Information for details). After 5 days, animals without dark urine were discarded and the remaining animals were randomly allocated to cages for CIH exposure protocol until day 28 after ligation.
All groups of rats were fed standard rat chow and were provided with drinking water ad libitum during the entire protocol. Rats were weighed before and after 14 days of exposure to CIH or normoxia. After the hemodynamic studies, the livers and spleens were weighed. Liver tissue samples were collected and stored at −80°C for control, advanced cirrhosis, and CBDL rats without liver perfusion.
Chronic Intermittent Hypoxia Exposure Protocol
Rats were maintained on a 12-hour light/dark cycle and exposed to CIH for 12 hours/day during their diurnal sleep period for a minimum of 14 days. Using a time-programmed solenoid valve, nitrogen (98%) was distributed to the hypoxic chamber for 30 seconds at a constant flow that reduced the ambient fractional concentration of oxygen to 8%-10%. This fractional concentration of oxygen was maintained at this level for 60 seconds before an electric fan located at one side of the cage allowed a gradual return over 60 seconds to 20%-21% of oxygen for 60 seconds (Fig. 1). Regular checks of chamber oxygen concentrations during the experiment were made using an oxygen sensor (VMX300, Viamed, UK) and were registered through an amplifier and recorded on a computer data acquisition system (ADInstruments, Mountain View, LA).
For sham exposure (handled controls [HC]), rats were kept in an identical plastic cage placed side by side. The inflow gas was always room air, but the solenoid switches, fan, and inlets reproduced the noise and airflow disturbances of the CIH protocol.
Rats in both groups were housed six per cage in accordance with space recommendations in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1985). The temperature and the relative humidity of the chambers were maintained at 21%-24°C and between 30% and 70%, respectively. At the end of the 12-hour treatment period, animals were transferred to their standard cages in a separate area and housed under room air conditions for the remainder of the day in an animal care facility at the University of La Laguna. All protocols were approved by the Animal Care and Use Committee.
Hematocrit was evaluated in control (n = 46) and CCl4-induced advanced cirrhotic rats (n = 14) to confirm the effect of CIH. The blood samples (1-2 mL) were withdrawn from the tail, placed in microcapillary tubes, and spun in a microcentrifuge (MPW-250e, Med. Instruments, Warsaw, Poland) for hematocrit measurement.
In Vivo Hemodynamic Study
At the end of the intermittent hypoxia exposure protocol, rats were anesthetized as mentioned previously. First, a catheter was inserted in the carotid artery to monitor blood pressure (mean arterial pressure [MAP]; mm Hg) and heart rate (beats per minute, bpm), and into the portal vein through an ileocolic vein to measure portal pressure (mm Hg). The catheter was connected to a Power Lab (4SP) linked to a computer using Chart version 5.5.6 for Windows software (ADInstruments) and, after a period of 10 minutes of stabilization, recordings were performed with pressure transducers. In a supplementary group of cirrhotic rats, after baseline measurements following the stabilization period, HC and CIH rats underwent volume expansion with incremental doses (1-8 mL/kg, every 2 minutes) of hydroxyethyl starch 6% (Voluven 6% 130/0.4, Fresenius Kabi, Barcelona, Spain)16 via a femoral vein catheter.
Isolated Perfused Liver System
A flow-controlled perfusion system was used in this study. The system has been described elsewhere17 (see Supporting Information for details).
Baseline perfusion portal pressure (PP) was recorded before the intrahepatic microcirculation was preconstricted with the α1-adrenergic agonist methoxamine (Mtx, 10−4M) a well-characterized vasoconstrictor for the hepatic vasculature. Five minutes later, dose-response curves to cumulative doses of acetylcholine (ACh, 10−7, 10–6, and 10−5M) were evaluated. The concentration of ACh was increased by one log unit every 1.5 minutes. Response to cumulative doses of ACh was calculated as a percent change in PP.
In a different group of rats, a portal perfusion pressure-response curve to Mtx was obtained by adding increasing doses of Mtx (10−6, 10−5, 10−4, 5 − 10−4 mol/L) to the reservoir every 5 minutes.
Western Blot Analysis
Protein nitrotyrosination (3-NT), a marker of peroxynitrite production and oxidative stress due to NO reaction with ROS, was determined by western blotting (see Supporting Information for details).
Blots were probed with a mouse anti–3-NT (1:1,000) monoclonal antibody (Sigma, Madrid, Spain) and mouse anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA).
X-ray films were exposed, developed, fixed, and scanned. Densitometry of digital images was performed with Melanie version 6 software. GAPDH was used as control of sample loading.
Endothelial NO Synthase and Phosphorylated Endothelial NO Synthase.
Endothelial NO synthase (eNOS) and phosphorylated eNOS (p-eNOS) protein detection was performed with mouse anti-eNOS (1 μg/mL dilution; BD Biosciences, San Jose, CA) and rabbit anti–p-eNOS (1:500 dilution; Cell Signaling Technology) as described for 3-NT. Quantitative densitometric values were compared between eNOS and p-eNOS blots. GAPDH was used as control of sample loading.
Nitric Oxide Bioavailability.
Measurements of guanosine 3',5'-cyclic monophosphate, a marker of NO bioavailability, were performed in control and cirrhotic rat liver homogenates from HC and CIH rats (see Supporting Information for details). The results are expressed as picomoles per milliliter.
Drugs and Reagents
Mtx and ACh were purchased from Sigma (Madrid, Spain).
Statistical analysis was performed using SPSS version 15.0 for Windows (SPSS Inc., Chicago, IL). All data are reported as the mean ± SEM. Comparisons between groups were performed using analysis of variance followed by Student's t test or the nonparametric test for unpaired data (Mann-Whitney) when appropriate. Differences were considered significant at P < 0.05.
Rats after 12 weeks of CCl4 inhalation and CBDL rats had macroscopic cirrhosis and signs of portal hypertension as shown by the presence of ascites, collateral circulation, or splenomegaly (Table 1). Rats with 8 weeks of CCl4 inhalation showed macroscopic micronodular cirrhosis without ascites.
Table 1. Physiological Parameters in Control and Cirrhotic Rats Exposed to CIH or HC
Body weight was recorded to determine whether the exposure protocol altered weight gain. Baseline body weights were not different between CIH and HC rats, in the four groups at the beginning of the protocol (Table 1).
Control, CCl4, and CBDL rats increased in body weight between day 0 and day 14, but this was significantly lower only in control rats exposed to CIH compared with HC rats (Table 1). CBDL rats showed a trend either as an absolute or as a percentage increase (P = 0.11). No significant differences in liver weight were observed in control or cirrhotic rats.
Control rats exposed to CIH exhibited a significantly greater hematocrit compared with HC rats (56.7 ± 1.4 versus 51.7 ± 1.3; P ≤ 0.01). There was no significant difference in hematocrit in CIH and HC cirrhotic rats (47.4 ± 1.1 versus 45.4 ± 0.9), although it was significantly lower than in control rats (P < 0.05).
In Vivo Studies.
After 14 days of CIH protocol, there were no significant differences in MAP (P = 0.9) or heart rate (P = 0.5) measured in CIH and HC control rats, respectively (Table 2).
Table 2. Hemodynamic Parameters in Control and Cirrhotic Rats Exposed to CIH or HC
MAP (mm Hg)
Heart Rate (bpm)
In Vivo PP (mm Hg)
PP (mm Hg)
In vivo PP measurements were available in 6 CIH and 5 HC control rats. Data are presented as the mean ± SEM.
MAP was lower in early (P < 0.05) and advanced CCl4 and CBDL (P ≤ 0.01) cirrhotic rats compared with control rats. However, there were no differences between CIH and HC rats within the groups (Table 2). Heart rates were also nonsignificantly different.
Baseline values of PP were similar within the groups, but higher in all cirrhotic rats compared with control rats (Table 2). As expected, sequential volume expansion increased both MAP and PP in the three cirrhotic groups evaluated. However, CIH cirrhotic rats showed a lower MAP increase compared with HC (P = 0.06). Thus, a similar PP increase response was observed in CIH and HC rats (Fig. 2).
Liver Perfusion Studies.
As expected, cirrhotic livers showed a higher baseline portal perfusion pressure than control livers (P ≤ 0.01) (Table 2). However, there were no significant differences in baseline perfusion pressure between CIH and HC rats.
Control livers exhibited an incremental vasorelaxation in response to cumulative doses of ACh, whereas all cirrhotic rats showed less vasodilation or even paradoxical vasoconstriction (Fig. 3). CIH had no effect on dose-response curves to ACh in control rats (Fig. 3A). However, in livers from rats with advanced CCl4-induced cirrhosis, CIH exposure clearly and significantly attenuated the vasorelaxation in response to cumulative doses of ACh showing higher paradoxical vasoconstriction at the last dose (maximum at 10−5M: 48.7 ± 2.6 versus 23.9 ± 3.4% in HC; P ≤ 0.01) (Fig. 3D). However, CIH effects were less patent in CBDL and rats with early cirrhosis in our experimental setting, although a trend was still observed (Fig. 3B,C).
Mtx produced a significant, dose-dependent increase in portal perfusion pressure in control and cirrhotic livers (Fig. 4). CIH exposure did not modify the PP response to Mtx in control livers (Fig. 4A). In contrast, this maneuver further exacerbated the effect of Mtx on PP in early (maximum at 10−4M: 12.2 ± 1.5 versus 8.5 ± 1.1 mm Hg in HC; P = 0.08) (Fig. 4C), advanced CCl4 cirrhotic rats (maximum at 5 × 10−5M: 20.8 ± 1.9 versus 15.8 ± 1.1 mm Hg in HC; P = 0.05) (Fig. 4D), and CBDL rats (maximum at 5 × 10−5M: 14.2 ± 1.4 versus 9.7 ± 1.1 mm Hg in HC; P = 0.04) (Fig. 4B).
Effects on Oxidative Stress, eNOS, p-eNOS, and NO Bioavailability
CCl4 and CBDL cirrhotic livers exhibited an increase of oxidative stress compared with control rats (Fig. 5A). CIH was associated with a higher degree of oxidative stress in the liver of control (Fig. 5B), CCl4 cirrhotic rats (Fig. 5C) and CBDL rats (Fig. 5D), as indicated by increased content of 3-NT. In control rats these results were associated with a significantly increase in eNOS phosphorylation (Fig. 6A), whereas cirrhotic rats exhibited similar eNOS and p-eNOS (Fig. 6B, 6C). As expected, hepatic guanosine 3',5'-cyclic monophosphate levels were significantly higher in HC control rats compared with HC cirrhotic rats (1.60 ± 0.92 versus 0.92 ± 0.14 pmol/mL, P < 0.05). However, there were no statistical differences between HC and CIH exposure in control rats (1.60 ± 0.23 versus 1.37 ± 0.26 pmol/mL, P = 0.5) under the experimental conditions tested, although a trend was observed in CCl4 cirrhotic rats (0.92 ± 0.14 versus 0.60 ± 0.2 pmol/mL, P = 0.2). These data suggest that CIH exposure induces oxidative stress and probably eNOS phosphorylation, which is impaired in cirrhotic rats provoking attenuation in vasodilation and hyperresponse to the vasoconstrictor Mtx.
We used a conventional rat model of CIH to study whether this well-known independent factor for systemic endothelial dysfunction could also provoke intrahepatic endothelial impairment in two different animal models of cirrhosis. Our study provided evidence that a short period of CIH exposure can influence the regulation of hepatic vascular tone in cirrhosis, exacerbating intrahepatic endothelial dysfunction. In addition, our results suggest that CIH effects are more relevant as portal hypertension develops.
The most relevant finding in our study is that the intrahepatic vasodilation response to ACh was significantly attenuated in CCl4 cirrhotic rats exposed to CIH compared with HC rats. This result is in keeping with previous experiments showing that CIH exposure exerts similar hemodynamic effects in other vessels.5 In addition, a hyperresponse to Mtx was also observed in CCl4 and CBDL cirrhotic rats, which is also in agreement with previous data.6 An increase in oxidative stress was also found after CIH. Hence, theoretically the net result of increased oxidative stress and decreased eNOS activity accounts for the significant alteration in the vascular reactivity of cirrhotic livers.
CIH (and OSAS), in addition to causing systemic hypertension,18 ischemic heart disease,19 and stroke,20 has organ-specific effects in the lung leading to pulmonary hypertension.21 In all these disorders, endothelial dysfunction has been identified as an early event. Comparable to patients with OSAS, patients with cirrhosis also exhibit attenuation in vasodilation in response to ACh and hyperresponse to vasoconstrictors,2 which are hemodynamic abnormalities associated with intrahepatic endothelial dysfunction. In this case, a reduced NO bioavailability in the cirrhotic liver has been well demonstrated in relation with decreased eNOS activity22, 23 and to increased scavenging of NO by ROS.24, 25
Similar mechanisms have been suggested to be involved in CIH and systemic endothelial dysfunction. In particular, ROS production leading to NO scavenging has been implicated. In this regard, several recent studies have provided evidence for increased ROS generation in different tissues after exposure to repetitive episodes of hypoxia/reoxygenation.26 This occurs not only in vascular territories but also in the lung, heart, and brain.27, 28 In addition, endothelial dysfunction has been shown to improve after antioxidant administration.29 Our finding of increased nitrotyrosinated proteins in the liver after CIH exposure strongly supports the notion that OSAS and oxidative stress also target the liver with important hemodynamic effects.
In our study, despite the increase of nitrotyrosine proteins found in control rats after CIH, the intrahepatic vascular response was not different compared with HC rats. This could be due to compensatory feedback regulation of NOS expression30 in the healthy organ to provide sufficient local NO. In fact, in our setting, eNOS phosphorylation was found to be up-regulated after CIH. By contrast, cirrhotic rats exposed to CIH exhibited attenuation in vasodilation and increased levels of oxidative stress but no increase in p-eNOS and lower NO, which is clearly insufficient to achieve a balance. In this regard, it is well known that eNOS activity is already diminished in cirrhosis22, 23 and eNOS uncoupling, due to limited availability of cofactors required for NO production, promotes more superoxide, aggravating the lack of NO.17 It is conceivable then that ROS-mediated reduction of NO availability may not be compensated in cirrhosis and could contribute to the intrahepatic vascular responses observed.
In rats with early cirrhosis, hemodynamic effects after CIH exposure were less obvious. Surprisingly, vasodilation responses in the CBDL cirrhotic rats differed from those obtained in advanced cirrhotic CCl4 rats. Several reasons may account for these differences. First, the CBDL model provokes intense fibrotic presinusoidal portal hypertension,31 which may hinder the interpretation of results when assessing ACh responses related to the endothelial sinusoidal cell, even more so when the degree of relaxation/paradoxical vasoconstriction and the margin for differences is extremely low. Second, bilirubin has potent antioxidant properties and may be a contributing factor partially protecting the endothelium from CIH. In fact, antioxidants have shown to attenuate portal hypertension.24 Finally, and most importantly, hepatopulmonary syndrome develops in the majority of rats after CBDL.32 Interestingly, Imamura et al.33 demonstrated that CBDL cirrhotic rats were not impacted by chronic hypobaric hypoxia in terms of pulmonary hypertension as much as hypoxic healthy rats. These results were associated with increased expression of endothelin-1 and its receptor, together with e-NOS up-regulation as potential mechanisms of protection. Taking into account these experiments, it is plausible that CIH effects on vascular reactivity could be attenuated in the CBDL model, such as in sustained chronic hypoxia. On the other hand, further vasoconstriction to Mtx was observed in both models of cirrhosis after CIH. Our results suggest that additional factors may play a role in this response. Particularly, increased production of endothelin-1 has been found to occur during CIH.34
To our knowledge, this is the first experimental study investigating the hepatic hemodynamic effects of CIH in the setting of cirrhosis. Our novel findings are clinically relevant, because CIH and OSAS have been described in patients with cirrhosis and portal hypertension. A pilot study showed a previously undescribed high prevalence of OSAS and nocturnal oxygen desaturations among patients who have cirrhosis with ascites that improved after paracenthesis.10 This observation has been confirmed by other groups more recently.11, 13 The results of these studies showed that OSAS can be present in cirrhotic patients and particularly in those with severe liver disease, which could exacerbate impairment of liver function. In fact, OSAS has been associated with elevated alanine aminotransferase levels in patients12 and animals exposed to CIH.35 Furthermore, even severe histology changes (inflammation and fibrosis) have been shown to appear after long exposure to CIH.35 In our short-term experimental conditions, the absence of change in baseline portal perfusion pressure makes a change in intrahepatic mechanical vascular resistance unlikely due to increased fibrosis.
In vivo baseline hemodynamic parameters were not significantly different between CIH and HC rats. However, after volume expansion was performed in cirrhotic rats, analysis of hemodynamics yielded interesting results. As shown by other investigators,16, 36 after volume expansion in cirrhotic rats, PP increases as MAP and portal blood flow augments, due to the inability of the liver circulation to appropriately dilate in response to flow. In fact, this further increase in PP can be prevented with NO donors16, 36 without modifying MAP or portal blood flow. In our study, PP increase was similar in CIH and HC rats. However, MAP and probably PBF increase were lower in CIH rats. Indeed, vascular hyporeactivity due to autonomic impairment has been described recently after exposure to CIH.37 These observations suggest that CIH may also provoke additional deleterious systemic effects in cirrhotic rats, yet to be studied.
Overall, these data suggest that CIH could be a relevant underestimated factor to take into account when assessing cirrhotic patients with portal hypertension. In addition, such a possibility could be therapeutically relevant considering the beneficial impact of therapy for OSAS on hemodynamic responses3 and cardiovascular disorders, specifically systemic hypertension.38 Finally, OSAS is an increasingly common disorder in well-developed countries, frequently associated with obesity, leading to nighttime CIH. Interestingly, a recent subanalysis from a well-defined cohort of patients who had cirrhosis with portal hypertension identified obesity as an independent risk factor of clinical decompensation.39 Whether vascular endothelial alterations that are attributed to obesity may in fact be partially related to OSAS remains to be investigated.
In keeping with other published data, we also confirmed other effects of CIH. First, we observed a lower increase in body weight, as reported before,40 which has been related to leptin regulation.41 However, the liver weight was not different within groups, and a theoretical putative effect on the hemodynamic response can be dismissed. We also found that CIH rats exhibited a significant increase in hematocrit, which is also a well-described effect of sustained and intermittent hypoxia due to increased erythropoietic response.21, 40, 42 Nevertheless, hematocrit increase was not statistically significant in cirrhotic rats, probably due to hemodilution, ineffective erythropoiesis, and splenomegaly. A similar observation has been reported after sustained chronic hypoxia in CBDL rats.33
Systemic blood pressure has been shown to increase in animals after long-term exposure to CIH.43 However, despite the increase in hematocrit, which may enhance vascular resistance by increasing blood viscosity, systemic blood pressure was not found to be significantly elevated in our setting. Differences in the strain of rats used in the studies and the degree and duration of hypoxic exposure might explain the differences. Concerning this issue, others using an identical CIH protocol had results similar to ours.5 In addition, the absence of systemic blood pressure increase in such a short period of exposure to CIH is not surprising, because endothelial dysfunction is well known to be an early event.
In conclusion, using this model mimicking the episodic hypoxemia of OSAS in humans, we demonstrated that CIH exposure further exacerbates endothelial dysfunction that occurs in cirrhotic rats. This occurs together with increased oxidative stress, which may influence NO bioavailability. Our results provide a rationale to conduct clinical studies to assess whether OSAS exacerbates endothelial impairment in patients with cirrhosis.
We thank M. R. Arnau for animal care; M. C. Hernández and J. Abreu for technical expert assistance in OSAS; H. García and J. Gracia for technical help with nitrotyrosine and p-eNOS detection; V. Febles for assistance with the hypoxia chambers; Laboratorios Glez-Santiago for funding; and Fundación para la Investigación Biomédica Rafael y Clavijo for editorial support.