The authors have declared that no competing interests exist.
Pulmonary arterial hypertension (PAH) is a lethal disease and improved therapeutic strategies are needed. Increased pulmonary arterial pressure, due to vasoconstriction and vascular remodeling, causes right ventricle (RV) failure and death in patients. The treatment of Sprague-Dawley rats with SU5416 injection and exposure to chronic hypoxia for three weeks followed by maintenance in normoxia promote progressive and severe PAH with pathologic features that resemble human PAH. At 5–17 weeks after the SU5416 injection, PAH is developed with pulmonary vascular remodeling as well as RV hypertrophy and fibrosis. The present study investigated subsequent events that occur in these PAH animals.
At 35 weeks after the SU5416 injection, rats still maintained high RV pressure, but pulmonary vascular remodeling was significantly reduced. Metabolomics analysis revealed that lungs of normal rats and rats from the 35-week time point had different metabolomics profiles. Despite the maintenance of high RV pressure, fibrosis was resolved at 35-weeks. Masson’s trichrome stain and Western blotting monitoring collagen 1 determined 12% fibrosis in the RV at 17-weeks, and this was decreased to 5% at 35-weeks. The level of myofibroblasts was elevated at 17-weeks and normalized at 35-weeks.
These results suggest that biological systems possess natural ways to resolve pulmonary and RV remodeling. The resolution of RV fibrosis appears to involve the reduction of myofibroblast-dependent collagen synthesis. Understanding these endogenous mechanisms should help improve therapeutic strategies to treat PAH and RV failure.
Pulmonary arterial hypertension (PAH) remains a fatal disease without a cure [
Despite RV failure being the major cause of death among PAH patients, RV pathophysiology is not well understood [
The treatment of rats with SU5416, an inhibitor of vascular endothelial growth factor (VEGF) receptor, and chronic hypoxia develops severe PAH [
Male Sprague-Dawley rats (Charles River Laboratories International, Inc., Wilmington, MA, USA) were subcutaneously injected with SU5416 (20 mg/kg body weight; TOCRIS, Minneapolis, MN, USA), maintained in hypoxia for three weeks [
At the end of the experiments, some rats were anesthetized with the intraperitoneal injection of xylazine (10 mg/kg body weight) and ketamine (100 mg/kg body weight). They were then intubated and mechanically ventilated with a volume-controlled Inspira Advanced Safety Ventilator (Harvard Apparatus, Holliston, MA, USA). Rats were maintained on a heat pad and the temperature was kept at 37°C by using a TR-200 Temperature Controller connected to a rectal probe (Fine Scientific Tools, North Vancouver, Canada). After a thoracotomy, a Millar catheter (1.4 F) was inserted into the RV apex. RV pressure signals were recorded by using PowerLab with Chart 5 software (ADInstruments, Colorado Springs, CO, USA). Animals were then euthanized by excising the heart and lung.
The Georgetown University Animal Care and Use Committee approved all animal experiments, and the investigation conformed to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. A total of 64 rats were used in this study.
Heart and lung tissues were immersed in buffered 10% formalin at room temperature, and were embedded in paraffin. Paraffin-embedded tissues were cut and mounted on glass slides. Tissue sections were subjected to hematoxylin and eosin (H&E) stain, Verhoeff-Van Gieson stain (VVG), Masson’s trichrome stain, and immunohistochemistry using α-smooth muscle actin (Abcam, Cambridge, UK) and periostin (MyBioSource, San Diego, CA).
Lung tissues were homogenized in 50% methanol containing internal standards and then centrifuged at 13,000 rpm for 10 min. An equal volume of chilled acetonitrile was added to each sample tube, and then was vortexed and incubated overnight at -20°C. Next, the tubes were again centrifuged at 13,000 rpm for 10 min at room temperature and the supernatant was transferred to fresh tubes and dried under vacuum. The dried metabolite mixture was resuspended in 100 μl of 50% methanol for mass spectrometry analysis.
Samples were injected onto a reverse-phase column using an Acquity ultra-performance liquid chromatography (UPLC) system (Waters Corporation, USA). Mass spectrometry was performed by using a Quadrupole-time-of-flight mass spectrometer operating in either negative or positive electrospray ionization mode with a capillary voltage of 3.2 kV and a sampling cone voltage of 35 V. Data were acquired in centroid mode with a mass range from 50 to 850 m/z for TOF-MS scanning.
RVs were homogenized and protein gel electrophoresis samples were prepared as previously described [
Means and standard errors were calculated. Comparisons between two groups were analyzed by using a two-tailed Student’s
Rats were injected with SU5416, exposed to hypoxia for 3 weeks and maintained in the normoxic condition for 2, 5, 14 or 32 weeks thereafter. We designated these rats to be 5-, 8-, 17- and 35-week time points, respectively (
SU5416-injected rats were subjected to 3 weeks in hypoxia and then maintained in normoxia for 2, 5, 14 or 32 weeks (5-, 8-, 17- and 35-wks time points, respectively). Animals were anaesthetized, ventilated and the chest opened. A Millar catheter was inserted into the RV apex and hemodynamic measurements were performed. (A) Flow diagram of the experiments. (B) Representative hemodynamic traces. (C) Means ± SEM of RVSP. (D) Means ± SEM of dP/dtmax. (E) Means ± SEM of the ratio of RV weight to LV + septum weight. *Significantly different from each other at
Representative hemodynamic traces are shown in
The H&E staining results (
SU5416-injected rats were subjected to 3 weeks in hypoxia and then maintained in normoxia for 2, 5, 14 or 32 weeks (5-, 8-, 17- and 35-wks time points, respectively). (A) Representative lung H&E stain results at x200 and x400 magnifications. (B) Means ± SEM of % pulmonary arterial wall thickness. (C) Means ± SEM of % of completely occluded pulmonary arterial vessels (30–100 μm diameter). **Significantly different from each other at
As shown in
SU5416-injected rats were subjected to 3 weeks in hypoxia and then maintained in normoxia for 14 or 32 weeks (17- and 35-wks time points, respectively). The large panels on the left are representative VVG stains at x100 magnification. Sections of different sizes of pulmonary arteries (50-150nm) are enlarged (x1000) on the right, showing VVG stain, Masson’s trichrome (MT) stain and immunohistochemistry of α-smooth muscle actin (αSMA).
By contrast, thickened pulmonary vessels in PAH animals were accompanied by overall changes in the lung structure including the loss of alveolar space, especially at 8 weeks. The lung sections of rats with PAH at the 17-week time point (
Masson’s trichrome stain demonstrated perivascular fibrosis (arrow in Panel B1-MT) and increased collagen fibers in the adventitial layer (Panel B2-MT). Immunohistochemistry revealed the hypertrophy and hyperplasia of smooth muscle with an increase in α-smooth muscle actin expression (arrows in Panels B1-SMA and B3-SMA) and small arteries with a thick muscularized intima (muscularization of small pulmonary vessels) (arrow in Panel B4-SMA).
These pathologic features were found to be normalized at 35 weeks.
We asked whether this apparent reversal of pulmonary vascular remodeling represents the normalization of the remodeled lung or conversion to a different entity. Global metabolites were analyzed in the lungs of control rats as well as PAH rats at the 8- and 35-week time points using mass spectrometry. Statistically significant differences at
SU5416-injected rats were subjected to 3 weeks in hypoxia and then maintained in normoxia for 5 weeks (8-wks time point) or 32 weeks (35-wks time point). Metabolomics analysis was performed in the lung tissues. Volcano plots (on the left) and Partial least squares Discriminant Analysis (PLS-DA; on the right) for (A) 8-weeks PAH (green circles) vs. normal control (red circles), (B) 8-weeks PAH (green circles) vs. 35-weeks PAH (red circles) and (C) 35-weeks PAH (green circles) vs. normal control (red circles) are shown. Dot volcano plots represent fold changes (FC) on the x-axis and the statistical significance in
The metabolomics analysis showed that 35-week PAH and control lungs were clearly different, indicating that the event is not a reversal process to the original entity but rather the conversion of the remodeled lung to another entity with reduced remodeling. Further, 35-week PAH lungs had 5.1-fold higher nicotinic acid, 3-fold lower retinoic acid, 2-fold lower cystine, 2-fold lower ascorbyl palmitate, 5.6-fold lower uric acid and 10-fold lower maleic acid/fumaric acid.
Complete metabolomics data are found in
The analysis of H&E-stained hearts of the control rats (
SU5416-injected rats were subjected to 3 weeks in hypoxia and then maintained in normoxia for 2, 5, 14 or 32 weeks (5-, 8-, 17- and 35-wks time points, respectively). (A) Representative H&E staining results of the RV tissue sections. (B) Masson’s trichrome stain results in the RVs. Blue stains indicate fibrotic areas. Bar graph represents means ± SEM of % fibrotic area (N = 7). *Significantly different from each other at
Cardiac fibrosis was promoted by SU5416/hypoxia treatment in the RVs at 5 weeks (to 7% fibrotic area), persisted at 8 weeks and further increased at 17 weeks to 12% fibrotic area (
The reduction of collagen expression at 35-week time point compared to 17-weeks could be due to decreased synthesis or increased degradation.
SU5416-injected rats were subjected to 3 weeks in hypoxia and then maintained in normoxia for 14 or 32 weeks (17- and 35-wks time points, respectively). Hearts were fixed in formalin and embedded in paraffin. Longitudinal sections of the RV myocardium were subjected to immunohistochemistry using the antibody against (A) α-smooth muscle actin and (B) periostin. Results are presented at x200 and x1,000 magnifications. Scale bars indicate 200 μm for x200 and 50 μm for x1,000. The brown stains indicate the expression of α-smooth muscle actin or periostin. Arrows depict that neither α-smooth muscle actin nor periostin stains are present in fibroblasts of the normal RV; brown α-smooth muscle actin and periostin stains are present overlapping nuclear stains of fibroblasts at 17-wks; and α-smooth muscle actin and periostin stains in fibroblasts are absent at 35-wks.
The major findings of the present study are that pulmonary vascular remodeling and RV remodeling can naturally be resolved in rats treated with SU5416 plus hypoxia. These findings have great translational importance because if the endogenous mechanisms of the repair/reversal of pulmonary arteries and RV remodeling are understood, therapeutic agents that mimic such natural pathways may be developed to help PAH patients. In this regard, the SU5416/hypoxia model in Sprague-Dawley rats could serve as a useful system to investigate these mechanisms.
While pinpointing the exact mechanisms through which remodeled pulmonary arteries and RV are repaired will need years of research, the present study suggested that reduced pulmonary remodeling is not due to the reversal to the original normal lung, but it seems that the lung has become a new entity with a distinct metabolomics profile. In this particular case, lung and pulmonary vascular structures appear normalized; however, RV pressure was found to be still elevated, suggesting that pulmonary vasoconstriction may be enhanced.
We further found that while cardiac fibrosis progressively increases from the 5- to 17-week time points in the RVs, these lesions are resolved at 35 weeks even though significantly increased RV pressure is still maintained. Thus, the reversal of RV fibrosis is not because the blood pressure decreased. This is a remarkable naturally-occurring process as we assessed that the degree of RV fibrosis that occurs in this model is as high as 12% of RV mass, indicating that the RV had lost >10% of the functional myocardium. However, the RV contractility, as measured by dP/dt, was not weakened. How fibrotic tissues are eliminated and what replaces fibrosis are key future questions. Since the overall RV mass should be reduced at 35 weeks because of the disappearance of fibrotic regions, it is likely that either hypertrophied already existing RV myocytes, newly generated RV myocytes or both fill the regions that were fibrotic.
The resolution of RV fibrosis, i.e. the reduction of the collagen expression seen at 35-week time point can be due to decreased collagen synthesis or increased collagen degradation. Immunohistochemistry showed that myofibroblasts (as stained with α-smooth muscle actin as well as periostin antibodies) are increased in the highly fibrotic RV compared to normal RV, but myofibroblast levels were normalized in association with the RV fibroblast resolution. Thus, it is likely that the mechanism of the RV fibroblast resolution involves the reduction of collagen-forming myofibroblasts.
This animal model is quite useful for studies of adaptive mechanisms, which occur in response to the remodeling of the lung and heart as discussed in this study. Further, by using this model, we have previously reported our findings that despite dramatic cardiac fibrosis and apoptosis occurring in remodeled RVs, RV contractility is maintained because of the generation of ‘super RV myocytes’ regulated by signaling through calsequestrin 2 [
In terms of translational utility, the limitation of this particular model using Sprague-Dawley rats treated with SU5416/hypoxia is that these rats do not die of RV failure; thus, one cannot perform drug studies to investigate the effectiveness on survival. For this purpose, Fischer rats treated with SU5416/hypoxia would be more useful, because these rats die in response to developing PAH [
(XLSX)
(XLSX)
(XLSX)
(XLSX)
(XLSX)
(XLSX)
(PPTX)