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Abstract
We assessed the pulmonary hemodynamic response to vascular endothelial growth factor receptor, type 2, inhibition using SU5416 (SU) with and without chronic hypoxia (CH) in different background strains and colonies of rats. A single subcutaneous injection of SU (20 mg/kg) or vehicle was administered to different substrains of Sprague-Dawley (SD) rats, and they were compared with Lewis and Fischer rats, with and without exposure to CH (10% O2 for 3 wk). Remarkably, a unique colony of SD rats from Charles River Laboratories, termed the SD-hyperresponsive type, exhibited severe pulmonary arterial hypertension (PAH) with SU alone, characterized by increased right ventricular systolic pressure, right ventricular/left ventricular plus septal weight ratio, and arteriolar occlusive lesions at 7–8 weeks (all P < 0.0001 versus vehicle). In contrast, the other SD substrain from Harlan Laboratories, termed SD-typical type, as well as Fischer rats, developed severe PAH only when exposed to SU and CH, whereas Lewis rats showed only a minimal response. All SD-typical type rats survived for up to 13 weeks after SU/CH, whereas SD-hyperresponsive type rats exhibited mortality after SU and SU/CH (35% and 50%, respectively) at 8 weeks. Fischer rats exposed to SU/CH exhibited the greatest mortality at 8 weeks (78%), beginning as early as 4 weeks after SU and preceded by right ventricle enlargement. Of note, a partial recovery of PAH after 8 weeks was observed in the SD-typical type substrain only. In conclusion, variation in strain, even between colonies of the same strain, has a remarkable influence on the nature and severity of the response to SU, consistent with an important role for genetic modifiers of the PAH phenotype.
Clinicians are often faced with patients who, despite exhibiting a similar severity of pulmonary hypertension, have marked differences in their functional status; for instance, one patient may be nearly asymptomatic and the other with World Health Organization class IV limitations. This manuscript explores differences in the response of rats from various strains and substrains to a standard stimulus for pulmonary arterial hypertension that better reproduces all the pathological features of the clinical disease. The finding that different genetic backgrounds can dramatically alter susceptibility to pulmonary arterial hypertension, as well as the ability of the right heart to adapt to hemodynamic abnormalities, is an important advance in defining the mechanisms that underlie the variability in presentation and progression of patients with this condition.
Pulmonary arterial hypertension (PAH) is a progressive and lethal disease characterized by pulmonary vascular dysfunction and arterial remodeling (1–3), eventually leading to widespread loss of pulmonary microvasculature, right ventricular heart failure, and premature death (4, 5). Although there have been significant advances in the treatment of PAH during the last decade, the diagnosis and prognosis remain poor. Existing animal models of pulmonary hypertension (PH), such as the monocrotaline or chronic hypoxia (CH) model, reproduce only certain aspects of the disease (6). Until recently, no animal PH model fully replicated the human PAH phenotype, which included the development of complex plexiform lesions (7–10), often regarded as the hallmark characteristic of severe human PAH (11).
The inhibition of vascular endothelial growth factor receptor, type 2 (VEGFR2), by a single injection of the receptor tyrosine kinase inhibitor, SU5416 (SU), results in severe PAH in rats when combined with 3 weeks of exposure to CH (5, 12, 13). The SU compound is believed to cause microvascular lung endothelial cell (EC) apoptosis, likely by the withdrawal of vascular endothelial growth factor receptor survival signaling. This is thought to result in the emergence of apoptosis-resistant and hyperproliferative vascular cells that contribute to the development of complex, occlusive arterial lesions (12, 14), which bear a remarkable similarity to the plexiform lesions seen in the lungs of patients with this disease, especially at the later stages of the disease model. To date, most studies in the SU/CH model have used Sprague-Dawley (SD) rats. However, it is not known whether there are important differences in the phenotype of severe PAH in other rat strains. Immunodeficient athymic nude rats have been reported to be more susceptible to SU and to develop severe PAH even in the absence of hypoxic exposure (15), which was attributed to the loss of regulatory T cells in this model. Recently, it has been reported that Fischer rats appear to exhibit greater mortality in response to SU and CH than do the more commonly used SD rats, even though there was no obvious difference in the hemodynamic changes (16); however, the underlying mechanism for this difference was not defined.
In this study, we carefully evaluated the response to SU in the presence and absence of CH in a number of commonly used strains of rats to explore the potential importance of variation in genetic background in the development of the severe PAH phenotype. We report marked differences in the severity and survival in the SU/CH model of PAH in SD compared with Lewis and Fischer rats and show, we believe for the first time, that a specific colony of immune-competent SD rats developed severe PAH even when exposed to SU alone.
All study protocols were approved by the animal ethics and research committee (University of Ottawa, Ottawa, Ontario, Canada) and were conducted according to guidelines from the Canadian Council for the Care of Laboratory Animals. Details regarding the sources of the animals and the experimental procedures are described in the online supplement.
All values are expressed as mean ± SEM. The differences among the multiple experimental groups were determined by one-way analysis of variance, followed by Tukey's post hoc analysis when an overall significant difference was found. Differences between two groups were analyzed using the Mann-Whitney test unless otherwise specified. Survival curves were compared using Log-rank Mantel-Cox analysis. The data are presented as mean ± SEM unless otherwise stated, and a P value of <0.05 was considered to be statistically significant.
The response to SU treatment alone in the SD background was critically dependent on the specific colony from which the rats were obtained. At 7–8 weeks after SU injection, SD rats obtained from the Charles River colony exhibited marked increases in pulmonary arterial pressure and right ventricle (RV) remodeling in response to SU alone (Figure 1) (P < 0.0001 versus vehicle control) and therefore are referred to as “SD-hyperresponsive type” (SDHT) rats. In contrast, SD rats obtained from Harlan Laboratories showed no response to SU alone, which is typical for this background strain and therefore are referred to as “SD-typical type” (SDTT) rats. Of note, SDHT rats exhibited a bimodal response to SU alone (see Figure E1A in the online supplement); a clear increase in right ventricular systolic pressure (RVSP) was observed in 13 of the 18 rats studied (72%), whereas there was little or no response in the remaining 5 SDHT animals. A similar bimodal trend was seen for RV remodeling as well (Figure E1B). The combination of SU and CH did not produce any further increase in RVSP or RV hypertrophy in SDHT rats compared with SU alone (Figures 1A and 1B) but did increase the consistency of the response for hemodynamic changes and RV remodeling (Figures E1A and E1B). In contrast, SDTT and Fischer rats developed severe PAH only in response to SU together with CH (Figures 1A and 1B), and Lewis rats exhibited only modest PAH in response to SU/CH.
Figure 1. (A) Right ventricular systolic pressure (RVSP) and (B) RV remodeling right ventricle/left ventricle + septum (RV/LV + S) at 8 weeks after treatment with SU or vehicle (Veh), with or without a 3-week exposure to chronic hypoxia (CH). Data are shown for Sprague-Dawley hyperresponsive type (SDHT) (12–18/group) or Sprague-Dawley typical type (SDTT) (3–6/group), Fischer (6–14/group), and Lewis (4–8/group) rats. ***P < 0.001 compared with Veh; ###P < 0.001 compared with CH alone. RV, right ventricle; SU, SU5416.
[More] [Minimize]Histological analysis of H&E-stained lung sections from SDHT rats at 7–8 weeks after SU alone showed evidence of complex pulmonary arteriopathy, including occlusive plexiform-like lesions (Figure 2A), with similar numbers of occlusive lesions in rats treated with SU alone or with SU/CH (Figure 2B). SDTT and Fischer rats demonstrated complex lesions only in response to SU/CH, with a similar number of occlusive vascular lesions compared with SDHT rats in the presence or absence of CH (Figures 2C and 2D). In contrast, no occlusive lesions were seen in Lewis rats in the SU/CH group (Figure 2E). Similarly, the increased pulmonary arterial wall thickness was similar in SDHT rats exposed to SU alone and SU/CH (Figures E4A and E4B), whereas arterial remodeling in SDTT rats was seen only in response to SU and CH. Moreover, the degree of wall thickening was similar in all backgrounds exposed to SU/CH, with the exception of Lewis rats (Figures E4C and E4D).
Figure 2. (A) Representative microscopy images demonstrating the presence of complex, plexiform-like occlusive lesions in the pulmonary vasculature in SDHT rats treated with 0.5% carboxymethylcellulose sodium (CMC) (vehicle control) or SU. (B) Quantitative analysis of lung sections showed that occlusive lesions were detected in SDHT rats exposed to either SU alone or SU and CH. In contrast, (C) SDTT and (D) Fischer rats exhibited complex lesions only when exposed to both SU and CH. No occlusive pulmonary vascular lesions were observed in Lewis rats in the SU/CH group. Scale bar represents 50 μm. * and **P < 0.05 and 0.01, respectively, compared with vehicle-treated rats. LPF, lesions per field.
[More] [Minimize]Caspase-3 activation was assessed in the lungs of SDHT and SDTT rats at 1 week after treatment with SU alone. Increased cleaved caspase-3 immunostaining was seen in lung sections from SDHT rats, which was associated with arteriolar endothelium as seen in adjacent sections stained with an endothelial marker, vWF (Figure E3A). Increased cleaved caspase-3 in lung lysates from SDHT rats compared with SDTT rats was confirmed by immunoblotting (Figures E3B and E3C).
Mortality at 8 weeks after SU injection was markedly different in the four different strains of rats. SDHT rats treated with SU exhibited a significant mortality without or with CH (31% and 50%, respectively; P < 0.05 [Figures 3A and 4B]). In contrast, there was no mortality in SDTT and Lewis rats treated with SU or SU/CH. Mortality was the highest in Fischer rats exposed to SU/CH (77%; P < 0.01).
Figure 3. Kaplan-Meier survival curves in SDHT, SDTT, Fischer, and Lewis rats treated with (A) SU alone or (B) SU/CH. In the SU-alone group, only SDHT rats exhibited mortality (∼30%) over the 8-week study. *P < 0.05 versus Fischer using log-rank (Mantel-Cox) test. However, in SU/CH-treated rats, substantial mortality was seen in both SDHT (∼50%) and Fischer (∼80%) rats, but still no mortality was observed in SDTT and Lewis rats, with all animals surviving to 8 weeks. *P < 0.05 versus SDTT; **P < 0.01 versus SDTT. #P < 0.05 versus SDHT using log-rank (Mantel-Cox) test.
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Figure 4. Serial echocardiography measurements in SDTT versus Fischer rats. (A) Representative images illustrating increased RV dilatation in Fischer compared with SDTT rats in response to SU/CH. (B) The ratio of end-diastolic right ventricle to left ventricle internal diameter (RVID-d/LVID-d) was moderately increased in response to SU/CH in survivor (S) SDTT and Fischer rats compared with controls (Ctrls). However, there was a further marked increase in RV dilatation in nonsurvivor (NS) Fischer rats. *P < 0.05 versus Ctrl; **P < 0.01 versus Ctrl; ***P < 0.001 versus Ctrl; ##P < 0.01 NS versus S.
[More] [Minimize]Serial echocardiography was performed at regular intervals in the SU/CH model, and RV adaptation was assessed by measuring the RV relative to the left ventricle internal diameter in the diastole (RVID-d and LVID-d, respectively). Figure 4A shows representative two-dimensional echocardiographic images of SDTT and Fischer rats under control conditions and in the SU/CH model of severe PAH. Overall, there was a strong trend toward a greater increase in RVID-d/LVID-d in Fischer compared with SDTT rats (P = 0.06) in the severe PAH model, regardless of whether or not the rats survived to the end of the study at 8 weeks (Figure 4D). However, the RV diameter during the final week of life was markedly increased in the nonsurviving Fischer rats compared with the surviving Fischer (P < 0.01) or SDTT rats (P < 0.005), consistent with a strain-specific failure of RV adaptation in response to severe PAH in these animals.
Because SDTT rats showed no mortality at 8 weeks despite exhibiting severe PAH, a separate cohort was followed for more than 13 weeks after SU injection with or without CH. As before, the severe PAH phenotype was seen in animals treated with both SU and CH, and still no mortality was evident even at 13 weeks. Interestingly, not only was there no further increase in RVSP and RV hypertrophy, but RVSP and RV/left ventricle + septum at 13 weeks were significantly lower than at 8 weeks; this was also associated with a reduction in the number of occluded vessels at 13 weeks compared with 8 weeks after SU/CH (Figure 5A), consistent with possible resolution of occlusive arterial remodeling at late time points. Additionally, there was a direct correlation between RVSP and the number of occlusive lesions in these animals (Figure E2). To confirm an improvement in the severe PAH phenotype in this background at later time points, serial echocardiography was performed at 0, 3, 4, 8, and 13 weeks in both the SDTT and the SDHT substrains in the SU/CH model (Figure 5B). In SDTT rats, the maximal decrease in pulmonary artery acceleration time (PAAT) was seen at 3 weeks of CH. There was a substantial initial recovery in PAAT (∼30%) in the first week after return to normoxia (Figure 5C), and further improvement was seen between 8 and 13 weeks, yielding a total hemodynamic recovery of almost 75%. In contrast, SDHT rats showed no significant improvement in pulmonary hemodynamics either on return to normoxia or for up to 8 weeks. Later time points could not be assessed in this substrain because of the high mortality beyond 8 weeks.
Figure 5. Spontaneous recovery from the severe pulmonary arterial hypertension phenotype in SDTT rats. (A) Summary data for RVSP, RV/LV + S, and occlusive lesions at 8 weeks and 13 weeks after SU/CH exposure. (B) Representative images showing the echocardiographic measurement of pulmonary artery acceleration time (PAAT) at 8 weeks in SDHT and SDTT rats. (C) Summary data showing serial echocardiography demonstrating the time course of changes in PAAT or PAAT recovery ratio measured over 14 weeks in SDHT (n = 4) and SDTT (n = 5) rats treated with SU/CH. PAAT recovery ratio was calculated as the difference between the PAAT at the time point shown and the peak reduction in PAAT at 3 weeks, divided by PAAT at 3 weeks. *P < 0.05, 8 versus 13 weeks; ¶P < 0.1, 8 versus 13 weeks; ##P < 0.01 versus 3 weeks by paired t test.
[More] [Minimize]We evaluated the response to SU in the presence and absence of CH in different rat strains that have been used widely in experimental PH studies (5, 17–19) to better understand the importance of genetic background in the development of a severe PAH phenotype. We now report that even minor differences in background strain have a remarkable influence on both the severity and the nature of the phenotype in the SU/CH model of severe PAH.
The development of a model of severe PAH that better reflects the pathological features of the human disease, in particular the development of complex intimal and plexiform-like lesions (11), has been an important advance. Taraseviciene-Stewart and colleagues were the first to report that inhibition of VEGFR2 using a receptor tyrosine kinase inhibitor, SU, accentuated the hemodynamic response to CH in SD rats and resulted in the appearance of intimal and complex arteriolar lesions (12). This was associated with a marked increase in EC apoptosis, and the effects of SU on both hemodynamics and vascular remodeling could be abrogated by nonspecific caspase inhibition, confirming a causal role of apoptosis in the severe PAH phenotype. This led to the concept that EC apoptosis triggered the emergence of apoptosis-resistant, hyperproliferative vascular cells that contributed to the formation of complex and occlusive pulmonary arteriolar lesions in PAH (20). Abe and colleagues later demonstrated that severe PAH in SD rats in response to SU/CH was associated with excellent long-term survival over more than 13 weeks. Although hemodynamic abnormalities were evident by 4 weeks in this study, complex arteriopathy was not observed before 8 weeks after the SU compound was received (5), prompting the authors to suggest that these lesions were a consequence rather than a cause of the hemodynamic abnormalities. Nonetheless, in the current study, we found a direct correlation between RVSP and the number of occlusive lesions in SD rats (Figure E1), which is suggestive of a causal link between these lesions and the hemodynamic abnormalities of PAH. Moreover, we have reported previously the appearance of complex arterial lesions in as early as 3 weeks in the severe PAH model (21), a time point at which the increased pulmonary pressures are first observed.
Although no single model can replicate all features of the human disease, the SU/CH rat model has now been adopted widely as the most relevant for the study of basic mechanisms and novel treatment approaches in PAH. However, less is known about the response to SU/CH in background strains of rats other than SD. It is not uncommon for variable phenotypes to be found in different background strains. Indeed, previous studies have reported marked differences in systemic arterial remodeling in various rat background strains after balloon injury (22) and in the severity of ischemic stroke even between rats of the same strain (SD) from different suppliers (23). In addition, the Voelkel group has reported previously that athymic nude rats showed increased susceptibility to SU, developing severe PAH even without exposure to CH (15). However, this trait was attributed to a disordered immune response caused by a loss of T-regulatory cell activity.
We first studied two different colonies of SD rats: one from Charles River Laboratories in Canada and another from Harlan Laboratories in the United States. “Hyperresponder-type” SD (SDHT) rats from the Canadian Charles River colony developed severe PAH, including the development of occlusive arteriolar lesions, after even a single injection of the SU in the absence of CH. In contrast, the SD “typical-type” (SDTT) rats from Harlan showed no response to SU alone and required both SU and CH to exhibit a PAH phenotype, as has been reported previously. Interestingly, the response of SDHT rats to SU alone was clearly biphasic, with ∼75% of animals exhibiting a severe PAH phenotype, whereas the rest showed no response. This “all or none” pattern of response suggests that susceptibility to SU could relate to genetic traits that confers responsiveness to SU alone. Indeed, we found that SDHT rats from the Canadian Charles River colony exhibited a marked increase in lung-activated caspase-3 in response to SU alone by both immunostaining and Western blotting, whereas there was no detectable increase caspase activity in the SDTT rats. Therefore, these animals are uniquely susceptible to inhibition of VEGFR2, and do not require a second hit, such as hypoxia, for EC injury and apoptosis to be manifested after treatment with SU. This is consistent with the possibility of mutations in the SDHT substrain in genes that may modify the activity of pathways involved in EC growth and survival. In contrast, Lewis rats were the least responsive, showing no significant hemodynamic abnormalities and only minimal RV remodeling in response to SU/CH and no evidence of complex vascular lesions. Such exquisite sensitivity to variation in strain and even colony supports the importance of genetic modifiers in determining both the susceptibility to SU and the severity of the PAH phenotype in response to SU and CH. It should be noted that SD represents an outbred strain, and this no doubt contributes to some of the variability in response to SU between different substrains and colonies.
Interestingly, SDTT rats obtained from Harlan Laboratories tolerated severe PAH following treatment with SU and CH remarkably well, with no mortality for up to 13 weeks. In contrast, Fischer rats exhibited a high mortality in the SU/CH model of severe PAH compared with SD rats, despite comparable severity of hemodynamic abnormalities, which was consistent with a previous report (16). We hypothesized that this marked difference in survival may be a result of strain-specific differences in the ability of the RV to adapt to chronic increases in afterload. Indeed, on serial echocardiographic assessments, RV dilatation was seen only in the Fischer rats that later showed mortality. This finding suggests that a defect in RV adaptation to the increase in afterload in severe PAH contributed to poor survival in the Fischer background strain. RV dysfunction is well recognized as the major determinant of poor outcome and death in patients with PAH (24, 25), and, therefore, this model provides a unique opportunity to explore both the mechanisms underlying RV failure in severe PH and to develop strategies aimed at improving RV adaptation in severe PH.
The SU/CH model has been considered to be a model of irreversible PAH (12). However, using continuous hemodynamic monitoring (17), it was recently reported that SU/CH-induced PAH was partially reversible in Charles River SD rats obtained from a German supplier, with a substantial reduction in RVSP occurring mainly after the return to normoxic conditions, despite progressive increases in occlusive intimal arterial remodeling. In our study, we also found a significant improvement in RVSP and RV remodeling from 8 to 13 weeks after SU/CH in SDTT rats, which, in contrast to the previous report, was associated with a reduction in the number of occlusive arterial lesions. Again, this is consistent with a tight relationship between hemodynamic abnormalities and arterial remodeling in this model, in both the development and the resolution of severe PAH. In addition, serial echocardiographic assessment confirmed improvement in pulmonary hemodynamics in the same animals over this period in the SU/CH model. In contrast, SDHT rats from the Canadian colony failed to show any improvement in PAAT on return to normoxia or over the next 5 weeks under conditions of normoxia. It is intriguing that such marked differences in reversibility were observed in our study compared with the previous report of de Raaf and colleagues despite using the same outbred strain of rats from the same supplier (i.e., Charles River) (17). These findings may once again point to the substrain-dependent differences in the severe PAH phenotype such that colonies from the same supplier may vary based on geographic location (Canada versus Germany).
We have shown that differences in background strain have a profound influence on the severity and nature of the PAH phenotype in response to SU. In particular, we have identified a specific colony of SD rats exhibiting hyperresponsiveness to SU alone. In addition to the obvious importance of this unique colony for the identification of potential genetic modifiers, the use of the SDHT background will allow wider access to a severe PAH model even in laboratories that do not have access to CH chambers. In contrast, “typical-type” SD rats show excellent survival in the SU/CH model and are well suited to the study of novel therapies of established PAH using a “treatment” protocol. In addition, the high mortality seen in Fischer rats likely reflects a strain-specific defect in RV adaptation in response to the increased hemodynamic load, and this background is uniquely well suited to study the mechanisms of RV remodeling, whereas Lewis rats were unresponsive to SU/CH and are not a suitable strain for this model of PAH. Thus, the current findings will be highly relevant in the selection of the appropriate background strain for future experimental studies using the SU/CH model, and also provide novel insights into the mechanisms underlying the development and adaptation to severe PAH in different rat strains.
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This work was supported by a grant from the Canadian Institutes of Health Research (57726). Additional funding was provided by an unrestricted grant from Northern Therapeutics Inc. and United Therapeutics, Corp.
Author Contributions: B.J., D.W.C., and D.J.S. contributed to the conception and design of the study; B.J., Y.D., and M.T. contributed to the acquisition of data; B.J., C.S., and D.J.S. contributed to the analysis and interpretation of the data; B.J., C.S., K.R.C., D.W.C., and D.J.S. contributed to the drafting of the manuscript and review for important intellection content.
This article has an online supplement, which is accessible from the issue’s table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1165/rcmb.2014-0488OC on August 20, 2015
Author disclosures are available with the text of this article at www.atsjournals.org.
