TY - JOUR
AU - Kourembanas, Stella
AB - Abstract Pulmonary arterial hypertension (PAH) remains a serious disease, and although current treatments may prolong and improve quality of life, search for novel and effective therapies is warranted. Using genetically modified mouse lines, we tested the ability of bone marrow-derived stromal cells (mesenchymal stem cells [MSCs]) to treat chronic hypoxia-induced PAH. Recipient mice were exposed for 5 weeks to normobaric hypoxia (8%–10% O2), MSC preparations were delivered through jugular vein injection and their effect on PAH was assessed after two additional weeks in hypoxia. Donor MSCs derived from wild-type (WT) mice or heme oxygenase-1 (HO-1) null mice (Hmox1KO) conferred partial protection from PAH when transplanted into WT or Hmox1KO recipients, whereas treatment with MSCs isolated from transgenic mice harboring a human HO-1 transgene under the control of surfactant protein C promoter (SH01 line) reversed established disease in WT recipients. SH01-MSC treatment of Hmox1KO animals, which develop right ventricular (RV) infarction under prolonged hypoxia, resulted in normal RV systolic pressure, significant reduction of RV hypertrophy and prevention of RV infarction. Donor MSCs isolated from a bitransgenic mouse line with doxycycline-inducible, lung-specific expression of HO-1 exhibited similar therapeutic efficacy only on doxycycline treatment of the recipients. In vitro experiments indicate that potential mechanisms of MSC action include modulation of hypoxia-induced lung inflammation and inhibition of smooth muscle cell proliferation. Cumulatively, our results demonstrate that MSCs ameliorate chronic hypoxia-induced PAH and their efficacy is highly augmented by lung-specific HO-1 expression in the transplanted cells, suggesting an interplay between HO-1-dependent and HO-1-independent protective pathways. Lung, Hypoxia, Mesenchymal stem cell, Transgenic mouse Introduction Pulmonary arterial hypertension (PAH) is characterized by arteriolar wall remodeling, elevated pulmonary artery pressure, and right ventricular hypertrophy (RVH). Although currently there is no cure for this disease, treatment has improved during the past decade, offering both relief from symptoms and prolonged survival. Improving treatment strategies requires a thorough understanding of the molecular mechanisms underlying the pathophysiology of PAH and progress in the field has been significant, albeit slow, given the multifactorial nature of the disease. Research investigating the prevention of PAH or the treatment of established disease using animal models has explored the application of elastase inhibitors [1], inhibition of tyrosine kinase [2], inhalation of carbon monoxide (CO) [3], and the transplantation of endothelial-like progenitor cells expressing endothelial nitric oxide synthase (eNOS) [4]. Bone marrow cells can generate diverse nonhematopoietic cell types [5] and their efficacy in repairing tissue injury has been assessed in a number of systems. Transplantation studies in rodents, using whole donor bone marrow, or the plastic-adherent cell fraction, termed marrow stromal cells, multipotent stromal cells, or mesenchymal stem cells (MSCs) have reported on the potential contribution of these multipotent cells in lung repair [6–8]. Initial studies in mice provided evidence for transdifferentiation of transplanted MSCs into alveolar epithelium, with bleomycin injury apparently enhancing MSC engraftment in the lung [9–11], although overall engraftment rates were subsequently revised to be significantly less than originally estimated. MSC treatment has also been reported to confer protection in rat models of PAH induced by monocrotaline [12–14] and other models of lung injury [10, 11, 15–17], and transdifferentiation of MSCs into airway epithelial cells has been suggested as a potential therapeutic application for cystic fibrosis [18]. We have recently reported that MSC treatment can prevent lung damage in a neonatal mouse model of hyperoxia-induced bronchopulmonary dysplasia (BPD) despite low overall levels of donor cell engraftment, and importantly, we found that the in vitro MSC secretome can also confer protection, indicating that the therapeutic action of MSCs involves a significant paracrine component [19]. The inducible heme oxygenase-1 isoform (HO-1) responds to multiple stressors, including hypoxia, hyperoxia, acidosis, shear stress, and reactive oxygen species [20–23], and HO-1 activity restores homeostasis in many pathologic states by exerting anti-inflammatory, antiapoptotic, or antiproliferative effects on diverse cell types [24]. By degrading the pro-oxidant heme and generating the antioxidant bilirubin, HO-1 protects against oxidative injury. The second enzymatic product, CO, stimulates guanylyl cyclase and increases intracellular levels of cGMP, an important regulator of vascular tone and smooth muscle cell proliferation. We have previously reported on the essential role of HO-1 in cardiovascular adaptation to hypoxic stress and its protective function against lung inflammation, pulmonary hypertension, and right ventricular failure, that is, upregulation of endogenous HO-1 can prevent hypoxia-induced PAH in the rat [25], and transgenic mice harboring a human HO-1 (hHO-1) transgene with lung epithelium-specific expression are protected from the development of hypoxia-induced elevation in pulmonary artery pressure, RVH, and vascular remodeling [26]. Significantly, HO-1 modulates the transient inflammatory response induced in the lung very early on hypoxic exposure, which triggers the molecular cascades leading to subsequent pathology [26–28]. HO-1 deficient (Hmox1KO) mice subjected to chronic hypoxia exhibit exaggerated dilatation of the right ventricle with organized mural thrombi, which is not observed in wild-type (WT) hypoxic animals [29], suggesting that Hmox1KO cardiomyocytes specifically develop a maladaptive response to hypoxia and subsequent pulmonary hypertension. Supplementing Hmox1KO animals with exogenous CO or bilirubin reveals distinct protective actions of these two HO-1 enzymatic products on the heart and pulmonary vasculature [30]. In this study, we combined the two protective approaches, MSC treatment and HO-1 overexpression, and systematically assessed the efficacy of genetically engineered MSCs on treating established PAH in a mouse model. We transplanted MSCs from WT donors or transgenic donors designed to overexpress a hHO-1 transgene in the lung epithelium into either WT or Hmox1KO recipients prior to or after 5 weeks of hypoxic exposure. We report that MSC treatment can both prevent and reverse PAH induced by chronic hypoxia, and the efficacy of this treatment is highly augmented if the donor MSCs have the ability to overexpress HO-1 in the lung epithelium, apparently on transdifferentiation. Although MSC treatment conferred protection in both WT and Hmox1KO recipients, only WT animals exhibited complete prevention or reversal of established disease, highlighting the important role of endogenous HO-1 activity in protecting the lung. The MSC protective functions augmented by lung epithelial HO-1 expression include modulation of inflammatory mediators at the onset of hypoxia and antiproliferative action on vascular smooth muscle cells (VSMCs). Materials and Methods MSC Isolation and Transgenic Animal Lines Bone marrow-derived MSCs were isolated from 6- to 8-week-old mice and their differentiation potential was verified prior to transplantation, as previously described [19]. Donor animals were either WT FVB/n or belonged to one of the following transgenic lines: animals with constitutive, lung-specific expression of the hHO-1 transgene (SH01 line) [26]; Hmox1KO (a strain harboring a disruption of the murine Hmox1 locus) [29]; or bitransgenic mice with doxycycline (Dox)-inducible, lung-specific HO-1 expression (CC77 line). The latter line was derived from a cross between a line harboring a hHO-1 transgene under the control of the tetracycline operator (E. Vergadi and M.S. Chang et al., manuscript submitted for publication) and a line expressing the reverse tetracycline transcriptional activator (rtTA) under the control of the Clara cell secretory protein (CCSP) gene promoter [31] (a kind gift of Dr. J.A. Whitsett). The hHO-1 transgene expression in line CC77 was induced by the addition of 1 mg/ml Dox in the drinking water. All animal protocols were approved by the Children's Hospital Boston Animal Care and Use Committee. Pulmonary Arteriolar Wall Remodeling Randomly selected vessels with a diameter up to 100 μm in 4 μm H&E-stained or α-smooth muscle actin (α-SMA)-stained sections were captured at ×400 magnification. Medial wall thickness was measured as previously described [26] using the NIH ImageJ Program (http://rsbweb.nih.gov/ij/) and expressed as an index of vessel diameter as follows: (2 × medial wall thickness)/external diameter. Smooth Muscle Cell Proliferation Assay All tissue culture media were from GIBCO/InVitrogen, (Carlsbad, CA, www.invitrogen.com) and serum from Hyclone/ThermoScientific (Waltham, MA, www.thermoscientific.com). Conditioned media from either MSCs or mouse lung fibroblasts (MLFs) were produced for 24 hours under serum-free conditions followed by differential centrifugation at 420g for 10 minutes and 12,000g for 30 minutes to discard cells and debris, respectively. Clarified conditioned media were concentrated by ultrafiltration using a 10-kDa filter unit, and protein concentration was measured by Bradford assay (Bio-Rad, Hercules, CA, www.bio-rad.com). For proliferation assay, primary rat pulmonary artery smooth muscle cells (PASMCs) were inoculated at a concentration of 2 × 103 cells per well on 96-well plates in 100 μl DMEM containing 5% fetal bovine serum (FBS) and incubated for 24 hours under standard culture conditions. After serum starvation for 2 days in DMEM containing 0.1% FBS, cells were treated with either vehicle, conditioned media from cultured MSCs (MSC-CM) or MLF-conditioned media (MLF-CM), for 30 minutes, then 5% FBS was added to each well. After 48 hours, cell proliferation reagent WST-1 (Roche, Indianapolis, IN, www.roche-applied-science.com), which is cleaved by mitochondrial dehydrogenases in metabolically active cells to form formazan dye, was directly applied to the cells followed by further incubation for 3 hours. The absorbance for solubilized dark red formazan was determined by using a microplate reader. Statistical Analysis All values are expressed as means ± SD. Comparison between different groups was performed by one-way analysis of variance followed by Tukey's multiple comparison test using GraphPad Prism (5.0) (GraphPad, LaJolla, CA, www.graphpad.com). Significance was considered at p values less than .05. See Supporting Information for expanded Materials and Methods including information on: MSC isolation and the hypoxia-induced PAH mouse model, quantitative polymerase chain reaction (qPCR) and sequence of the PCR primers, probes, and reaction conditions, Y-fluorescent in situ hybridization (FISH) and immunohistochemistry, and assessment of microvessel density. Results Transplantation of Genetically Engineered MSCs Reverses Hypoxia-Induced PAH in WT Mice To determine the efficacy of MSC treatment in reversing hypoxia-induced pulmonary hypertension and to assess the contribution of HO-1 activity in the process, we delivered MSCs through jugular vein injection to mice after 5 weeks of hypoxic exposure and measured hemodynamic, vascular, and immunologic parameters after an additional 2 weeks in hypoxia. At the end of the 7-week hypoxic exposure, WT mice receiving Phosphate buffered saline (PBS) or WT-MSCs exhibited elevated right ventricular systolic pressure (RVSP), whereas the RVSP of WT mice receiving SH01-MSCs was within the normal range (Fig. 1A). The Fulton's index, the ratio of right ventricle weight over the weight of the left ventricle plus the septum (RV/[LV+S]), an index of RVH, in mice receiving SH01-MSCs was significantly lower than that of the hypoxic controls receiving PBS and not significantly different from that of normoxic animals (Fig. 1B). Interestingly, the Fulton's index of animals receiving WT-MSCs showed significant improvement when compared with PBS recipients, suggesting that mechanisms inherent to MSCs can ameliorate RVH independently of the hHO-1 transgene overexpression. Nevertheless, the presence of the hHO1 transgene in SH01-MSCs was required for complete reversal of disease. 1 Open in new tabDownload slide MSC transplantation reverses established pulmonary arterial hypertension (PAH). RVSP and Fulton's index measurements are shown for recipient WT (A, B) or Hmox1KO mice (C, D). SH01-MSCs reversed RVSP elevations and RVH in WT recipients and significantly reduced PAH in Hmox1KO animals, whereas WT-MSCs had only a partial ameliorative effect. Individual animal data are represented by filled circles and group means by horizontal lines. Data were analyzed by one-way analysis of variance followed by Tukey's multiple comparison test. Abbreviations: LV+S, left ventricle plus septum; MSC, mesenchymal stem cell; PBS, phosphate buffered saline; RV, right ventricle; RVSP, right ventricle systolic pressure; WT, wild-type. 1 Open in new tabDownload slide MSC transplantation reverses established pulmonary arterial hypertension (PAH). RVSP and Fulton's index measurements are shown for recipient WT (A, B) or Hmox1KO mice (C, D). SH01-MSCs reversed RVSP elevations and RVH in WT recipients and significantly reduced PAH in Hmox1KO animals, whereas WT-MSCs had only a partial ameliorative effect. Individual animal data are represented by filled circles and group means by horizontal lines. Data were analyzed by one-way analysis of variance followed by Tukey's multiple comparison test. Abbreviations: LV+S, left ventricle plus septum; MSC, mesenchymal stem cell; PBS, phosphate buffered saline; RV, right ventricle; RVSP, right ventricle systolic pressure; WT, wild-type. As a control experiment, we transplanted WT mice with MLFs under the conditions described earlier. There was no improvement in RVSP or RVH development under chronic hypoxia (Supporting Information Fig. S1, panels A and B) in accord with the results obtained with PBS-treated animals. Endogenous HO-1 Expression Is Essential for Reversal of RVH by MSC Treatment On transplanting Hmox1KO animals, which develop more severe pulmonary and cardiovascular injuries under hypoxia [29, 30], we observed significantly elevated RVSP in PBS recipients, as expected. As in the case of WT recipients, RVSP of Hmox1KO mice treated with SH01-MSCs returned to the normal range, and although WT-MSCs improved RVSP levels, they were not as efficacious as SH01-MSC treatment (Fig. 1C). In contrast to WT recipients, the Fulton's index of Hmox1KO mice treated with either SH01-MSCs or WT-MSCs remained within the pathologic range despite a significant improvement compared with the PBS-treated controls, especially in the group treated with SH01-MSCs (Fig. 1D). Combined, these results suggest that the degree of amelioration of RVSP and RVH in hypoxic animals is associated with the ability of donor MSCs to overexpress the hHO-1 transgene in recipient lung as well as the presence of an intact Hmox1 locus (i.e., endogenous HO-1 activity) in the WT recipients. SH01-MSC Treatment Reverses Vascular Remodeling and Prevents RV Dilatation and Thrombus in HO-1-Deficient Animals Compared with normoxic controls, marked medial wall thickening in pulmonary arterioles was observed after chronic hypoxia in both WT (Fig. 2) and Hmox1KO animals (Supporting Information Fig. S2). This remodeling was reversed only with SH01-MSC treatment, as evidenced by H&E and α-SMA staining of the pulmonary arterioles in both the WT animals (Fig. 2A–2F), and the Hmox1KO mice (Supporting Information Fig. S2, panels A–D). To assess other possible vascular effects imparted by MSC treatment, we have quantified the number of blood vessels per microscope field and the percent area per field representing blood vessels. No difference was found in overall blood vessel density between SH01-MSC-treated and PBS-treated groups (data not shown). 2 Open in new tabDownload slide MSC transplantation reverses lung vascular remodeling in hypoxic wild-type (WT) recipients. Increased medial wall thickness is evident in representative pulmonary arterioles of hypoxic animals receiving PBS (B, E) compared with normoxic animals (A, D) and complete regression of medial hypertrophy is observed in animals that received SH01-MSCs (C, F). H&E staining (A–C) and α-SMA staining (D–F) of the corresponding treatment groups. (G): The medial wall thickness index for each group. At least three sections of each lung were used and vessels of comparable size were photographed and analyzed. Data are expressed as means ± SD. Statistical analysis as in Figure 1. Normoxia, 72 vessels from 4 WT-mice; PBS group, 71 vessels from six mice; and SH01-MSC group, 108 vessels from eight mice. Abbreviations: MSC, mesenchymal stem cell; PBS, phosphate buffered saline. 2 Open in new tabDownload slide MSC transplantation reverses lung vascular remodeling in hypoxic wild-type (WT) recipients. Increased medial wall thickness is evident in representative pulmonary arterioles of hypoxic animals receiving PBS (B, E) compared with normoxic animals (A, D) and complete regression of medial hypertrophy is observed in animals that received SH01-MSCs (C, F). H&E staining (A–C) and α-SMA staining (D–F) of the corresponding treatment groups. (G): The medial wall thickness index for each group. At least three sections of each lung were used and vessels of comparable size were photographed and analyzed. Data are expressed as means ± SD. Statistical analysis as in Figure 1. Normoxia, 72 vessels from 4 WT-mice; PBS group, 71 vessels from six mice; and SH01-MSC group, 108 vessels from eight mice. Abbreviations: MSC, mesenchymal stem cell; PBS, phosphate buffered saline. We have previously reported that hypoxic Hmox1KO mice manifest exaggerated maladaptive response to chronic hypoxia, presenting significant dilatation of the RV compared with hypoxic WT animals. In addition, a majority of Hmox1KO animals develop RV mural thrombus after 5 weeks of chronic hypoxic exposure [29]. In the current study, we observed a similar proportion of mural thrombus formation in Hmox1KO animals that received PBS and in some recipients of WT-MSCs, but, most significantly, we observed no thrombus in any of the animals receiving SH01-MSCs (Supporting Information Fig. S2, panel E), the latter also presenting a reduction of the hypoxia-induced RV dilatation. This prevention of thrombus formation, in the context of only partial reversal of Fulton's index (Fig. 1D) indicates that MSC treatment is able to efficiently reverse the RV injury precipitated by systemic hypoxia, a maladaptive response we have suggested to be at least partially independent of the increased pulmonary vascular resistance in Hmox1KO mice [30]. Enhancement of the Efficacy of MSC Treatment Requires Epithelial Specificity of hHO-1 Transgene Expression The increased efficacy of SH01-MSCs over WT-MSCs in reversing PAH implies that donor MSCs acquire the ability to express the hHO1 transgene by transdifferentiating into pneumocytes competent to activate the SP-C promoter. We sought to confirm this hypothesis by using MSC donors from an independent transgenic strain, CC77, in which the hHO1 transgene is under the control of a Dox-inducible system. In these bitransgenic mice, hHO1 expression conforms to the expression profile of the CCSP-promoter-rtTA transgene, specifically, not only the expected expression in Clara cells but also the apparent ectopic expression in type II alveolar epithelium [31]. In the presence of Dox, the hHO-1 expression profiles in the lungs of SH01 animals and the lungs of CC77 animals share significant overlap. CC77 animals in the absence of Dox supplementation behave as WT animals. CC77-MSCs were delivered to WT mice and animals were subsequently exposed to hypoxia for 2 weeks and supplied with Dox in the drinking water to induce the hHO1 transgene. Dox supplementation efficiently protected CC77-MSC recipients from both, increases in RVSP and RVH, whereas, in the absence of Dox, RVSP measurements were similar to PBS recipients and the degree of RVH, although improved, remained over the normal range (Fig. 3). Thus, in the absence of Dox, the efficacy of CC77-MSCs in preventing PAH paralleled the decreased efficacy of WT-MSCs in reversing disease after 5 weeks of hypoxia. These results independently validate the importance of hHO-1 expression in augmenting MSC treatment. Aware of the reports summarized in [32] that chronic administration of high dosages of Dox may affect pulmonary parameters in certain studies, we administered Dox to one of the PBS control groups and observed no effect of Dox treatment on hypoxia-induced PAH (data not shown). 3 Open in new tabDownload slide Conditional expression on hHO-1 augments MSC efficacy in preventing pulmonary arterial hypertension. (A): RV pressure measurements of WT mice treated with PBS, or transplanted with CC77-MSCs and then exposed to hypoxia for 2 weeks without Dox, or CC77-MSCs plus Dox in the drinking water. (B): Fulton's index measurements of the same hypoxic WT mice after corresponding treatment, as indicated. Abbreviations: Dox, doxycycline; LV+S, left ventricle plus septum; MSC, mesenchymal stem cell; PBS, phosphate buffered saline; RV, right ventricular; RVSP, right ventricle systolic pressure; WT, wild-type. 3 Open in new tabDownload slide Conditional expression on hHO-1 augments MSC efficacy in preventing pulmonary arterial hypertension. (A): RV pressure measurements of WT mice treated with PBS, or transplanted with CC77-MSCs and then exposed to hypoxia for 2 weeks without Dox, or CC77-MSCs plus Dox in the drinking water. (B): Fulton's index measurements of the same hypoxic WT mice after corresponding treatment, as indicated. Abbreviations: Dox, doxycycline; LV+S, left ventricle plus septum; MSC, mesenchymal stem cell; PBS, phosphate buffered saline; RV, right ventricular; RVSP, right ventricle systolic pressure; WT, wild-type. Our results indicate that transplantation of MSCs capable of expressing the hHO-1 transgene in either a constitutive (SH01) or inducible (CC77) manner in the lung epithelium can achieve complete prevention and reversal of established PAH in WT mice. This suggests that, although MSC transplantation per se can partially ameliorate PAH pathology, the ability of donor MSCs to express a lung-specific hHO-1 transgene can greatly augment their efficacy. Most relevant, as hHO-1 transgene activation is restricted to pulmonary epithelium in our model, transdifferentiation of donor cells is apparently obligatory for the increased efficacy to occur. Transplantation, Clearance, and Engraftment of Donor MSCs We examined the fate of transplanted MSCs in the recipient lung by assessing Y chromosome DNA levels through qPCR to trace male donor cells in female recipients. As reported by others [33, 34], a rapid clearance of donor MSCs from the lung was observed and this occurred independently of normoxic or hypoxic exposure of the recipients. Male donor cells represented approximately 0.05% of the total cells in the lung of female recipients 48 hours postinjection and within a week postinjection, that value decreased by two orders of magnitude (Fig. 4A). At 2 weeks postinjection, lungs from hypoxia-treated mice had retained sixfold more donor MSCs than lungs of normoxic mice (Fig. 4B). The observed difference is statistically significant, and it may indicate enhanced engraftment in the injured lung. Nevertheless, given the low number of retained donor cells, and pending the complete characterization of the pneumocytes that the donor cells may have transdifferentiated into, the physiologic significance remains to be elucidated. 4 Open in new tabDownload slide Levels of transplanted MSCs and hHO-1 mRNA in the recipient lung. Time course of Y chromosome levels after male MSC transplantation in female recipients (A) and comparison between normoxic and hypoxic lungs 14 days postinjection (B). Multiplex real-time polymerase chain reaction assays were performed using murine Y-chromosome and GAPDH-specific primers. The results of engraftment were expressed as ratio between Y-chromosome DNA and GAPDH DNA. (C): Time course of human HO-1 (hHO-1) transgene mRNA levels in whole lung of wild-type mice transplanted with SH01-MSCs. (D): Relative hHO-1 mRNA in recipient mouse lungs transplanted with CC77-MSCs after 7 days of hypoxia in the absence (−) or presence (+) of Dox treatment. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; hHO-1, human heme oxygenase-1; PBS, phosphate buffered saline. 4 Open in new tabDownload slide Levels of transplanted MSCs and hHO-1 mRNA in the recipient lung. Time course of Y chromosome levels after male MSC transplantation in female recipients (A) and comparison between normoxic and hypoxic lungs 14 days postinjection (B). Multiplex real-time polymerase chain reaction assays were performed using murine Y-chromosome and GAPDH-specific primers. The results of engraftment were expressed as ratio between Y-chromosome DNA and GAPDH DNA. (C): Time course of human HO-1 (hHO-1) transgene mRNA levels in whole lung of wild-type mice transplanted with SH01-MSCs. (D): Relative hHO-1 mRNA in recipient mouse lungs transplanted with CC77-MSCs after 7 days of hypoxia in the absence (−) or presence (+) of Dox treatment. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; hHO-1, human heme oxygenase-1; PBS, phosphate buffered saline. Expression levels of the hHO-1 transgene in the lungs of WT recipients, although readily detectable by qPCR, were assessed to be three to four orders of magnitude less than the levels present in the lungs of SH01 or CC77 animals supplemented with Dox, where the transgene is widely expressed in the entirety of alveolar epithelium. This is in accord with the low number of donor cells retained in the lung. Interestingly, when expression levels of the hHO-1 transgene, derived from lungs of animals transplanted with SH01-MSCs, were normalized to the levels of Y chromosome at each corresponding time point postinjection, a clear increase of hHO-1 transcriptional activity per resident donor cell was observed 7 days postinjection, possibly indicating a time-dependent transdifferentiation of donor SH01-MSCs into pneumocytes (Fig. 4C). When CC77-MSCs were transplanted, expression of the hHO-1 transgene on day 7 was dependent on the presence of Dox (Fig. 4D) independently buttressing the notion that hHO-1 was activated in pneumocytes. Overall, the above data indicate that although a small number of donor cells remain in the recipient lung, at least a fraction of these resident cells has acquired the ability to activate either the SP-C or the CCSP promoter through transdifferentiation. Transdifferentiation of Donor MSCs to Alveolar Type II Epithelium To detect the distribution of resident donor cells in the lung, we used FISH staining for Y chromosome sequences. Rare male donor cells were detectable in the recipient female lung (Supporting Information Fig. S3A, green) and specificity of the signal was verified by observing the same positions using a red fluorescence filter (Supporting Information Fig. S3A, red). In addition, serial confocal scanning at 0.3-μm increment indicated that the Y-FISH signal was indeed within the observed cell (Supporting Information Fig. S3B). Furthermore, using immunohistochemistry and confocal microscopy on the lungs of recipients injected with green fluorescent protein positive (GFP+) MSCs, we detected expression of SP-C, a type II pneumocyte-specific marker within resident GFP+ cells, indicating transdifferentiation of MSCs to type II alveolar epithelium (Fig. 5A, 5B). Using confocal microscopy, we merged a red-stained GFP+ cell (Fig. 5C) with green-stained SP-C granules from the same cell (Fig. 5D) generating a yellow signal (Fig. 5E) and demonstrating that granular SP-C expression is within the transplanted cell. In addition, we produced three-dimensional (3D) digital reconstructions from a series of confocal images taken at 0.2-μm increment in the Z-axis through the region of interest. The optical dissection and orthogonal reconstruction of the 3D confocal images further confirmed that the expression of type II pneumocyte-specific SPC was indeed within the transplanted GFP+ cell (Fig. 5F). 5 Open in new tabDownload slide Type II pneumocyte-specific SP-C expression in a GFP+ cell. (A): Black SP-C granules indicate endogenous Type II pneumocytes (arrowheads) in the lung of a PBS recipient. (B): Black SP-C granules in an endogenous type II pneumocyte (arrowhead) and a GFP+ cell, which appeared to have assumed type II pneumocyte morphology (arrow). A GFP+ cell was labeled with Alexa Fluor 594 (red in [C]), whereas SP-C granules were labeled with Alexa Fluor 488 (green spots in [D]) from the same cell (arrow). Confocal microscopic colocalization of SP-C expression within GFP+ cells was evident by merging of these serial dual fluorescence images to generate yellow spots (E). (F): Z-stack of 42 images with 0.2-μm increment (original magnification ×600). Red/magenta-framed panel to the right indicates optical dissection and orthogonal reconstruction of the selected cell along the vertical plane (indicated by a red line), whereas green/turquoise-blue-framed panel above indicates optical dissection and orthogonal reconstruction of the selected cell along the horizontal plane (indicated by a green line). In both cases, the yellow granular SP-C can be identified within the GFP+ cell. Abbreviations: GFP+, green fluorescent protein positive; PBS, phosphate buffered saline. 5 Open in new tabDownload slide Type II pneumocyte-specific SP-C expression in a GFP+ cell. (A): Black SP-C granules indicate endogenous Type II pneumocytes (arrowheads) in the lung of a PBS recipient. (B): Black SP-C granules in an endogenous type II pneumocyte (arrowhead) and a GFP+ cell, which appeared to have assumed type II pneumocyte morphology (arrow). A GFP+ cell was labeled with Alexa Fluor 594 (red in [C]), whereas SP-C granules were labeled with Alexa Fluor 488 (green spots in [D]) from the same cell (arrow). Confocal microscopic colocalization of SP-C expression within GFP+ cells was evident by merging of these serial dual fluorescence images to generate yellow spots (E). (F): Z-stack of 42 images with 0.2-μm increment (original magnification ×600). Red/magenta-framed panel to the right indicates optical dissection and orthogonal reconstruction of the selected cell along the vertical plane (indicated by a red line), whereas green/turquoise-blue-framed panel above indicates optical dissection and orthogonal reconstruction of the selected cell along the horizontal plane (indicated by a green line). In both cases, the yellow granular SP-C can be identified within the GFP+ cell. Abbreviations: GFP+, green fluorescent protein positive; PBS, phosphate buffered saline. The apparent phenotypic conversion of MSCs to lung epithelial type II pneumocytes together with the hHO-1 expression data (Fig. 4) would suggest activation of the SP-C promoter driving the hHO-1 transgene in donor MSCs derived from the SH01 line. Thus, transplanted MSCs represent a minor fraction of the total lung cell population, but some of these resident cells appear to have transdifferentiated into lung alveolar epithelium and significantly augment the efficacy of MSC treatment by overexpressing HO-1. Transplanted MSCs Modulate Hypoxia-Induced Lung Inflammation We have previously reported that hypoxia triggers an early inflammatory response in the lung and when this response is suppressed by the anti-inflammatory action of HO-1, vascular remodeling is suppressed and the animals are protected from hypoxia-induced PAH [26]. Delivery of SH01-MSCs, but not of control MLFs, suppressed the hypoxia-induced inflammation by inhibiting macrophage accumulation in the bronchoalveolar lavage fluid of transplanted animals (data not shown). We assessed whether HO-1 expression contributed to the anti-inflammatory actions of MSCs by quantifying lung cytokine mRNA levels in WT mice treated with CC77-MSCs and exposed to hypoxia for 4 days in the presence or absence of Dox supplementation (Fig. 6). In this early time period, hypoxia greatly increased the expression of the proinflammatory cytokines, CCL2 (monocyte chemoattractant protein-1 [MCP-1]) and interleukin (IL)-6 in the lung, and MSC treatment inhibited this inflammatory response. The expression of the anti-inflammatory cytokine, IL-10, was unaffected by hypoxia, but MSC treatment resulted in a dramatic increase of IL-10 expression. Expression of IL-1 receptor antagonist was also decreased sixfold by hypoxia, and MSC treatment ameliorated this suppression (data not shown). The immunomodulatory function of MSCs was independent of the induction of hHO-1 expression, however, in all cases there was a trend of hHO-1 expression augmenting MSC efficacy. For example, Dox treatment of hypoxic CC77-MSC recipients resulted in IL-6 levels not significantly different from normoxic controls and in the case of CCL2, there was a statistical significance between the −Dox and the +Dox CC77-MSC groups (Fig. 6). 6 Open in new tabDownload slide MSC transplantation modulates the hypoxia-induced lung inflammation. Cytokine mRNA levels for CCL2, IL-6, and IL-10 were measured by quantitative RT-PCR in lungs of animals transplanted with CC77-MSCs and exposed to hypoxia for 4 days in the presence or absence of Dox to induce human HO-1 transgene expression. Data expressed as means ± SD. n = 4–5 animals per condition. For CCL2, *, p < .001 versus normoxic group; †, p < .01 versus PBS-hypoxia; ††, p < .001 versus PBS-hypoxia; ‡, p < .05 versus CC77-MSC-Dox treatment group. For IL-6, *, p < .05 versus normoxic group; †, p < .05 versus PBS-hypoxia; ††, p < .001 versus PBS-hypoxia. For IL-10, *, p < .01 versus normoxic group; †, p < .05 versus PBS-hypoxia; ††, p < .001 versus PBS-hypoxia. Abbreviations: Dox, doxycycline; IL, interleukin; MSC, mesenchymal stem cell; PBS, phosphate buffered saline. 6 Open in new tabDownload slide MSC transplantation modulates the hypoxia-induced lung inflammation. Cytokine mRNA levels for CCL2, IL-6, and IL-10 were measured by quantitative RT-PCR in lungs of animals transplanted with CC77-MSCs and exposed to hypoxia for 4 days in the presence or absence of Dox to induce human HO-1 transgene expression. Data expressed as means ± SD. n = 4–5 animals per condition. For CCL2, *, p < .001 versus normoxic group; †, p < .01 versus PBS-hypoxia; ††, p < .001 versus PBS-hypoxia; ‡, p < .05 versus CC77-MSC-Dox treatment group. For IL-6, *, p < .05 versus normoxic group; †, p < .05 versus PBS-hypoxia; ††, p < .001 versus PBS-hypoxia. For IL-10, *, p < .01 versus normoxic group; †, p < .05 versus PBS-hypoxia; ††, p < .001 versus PBS-hypoxia. Abbreviations: Dox, doxycycline; IL, interleukin; MSC, mesenchymal stem cell; PBS, phosphate buffered saline. MSC Conditioned Media Inhibit Smooth Muscle Cell Proliferation Chronic hypoxia results in excess smooth muscle cell layers around the distal pulmonary arterioles. This remodeling contributes to enhanced vasoconstriction and elevations in pulmonary vascular resistance characteristic of PAH. As cultured VSMCs exposed to hypoxia manifest increased proliferation in response to a mitogenic stimulus [35] with increased lifespan [36] compared with normoxic controls, excess VSMC growth and survival may underlie the vascular remodeling of hypoxia-induced PAH. To investigate the paracrine effects of MSCs on smooth muscle cell proliferation, we treated PASMC cultures with MSC-CM and measured cell proliferation, using MLF-CM as controls. MSC-CM, but not MLF-CM, inhibited serum-induced smooth muscle cell proliferation (Fig. 7A), and the effect was dose dependent (Fig. 7B). These data demonstrate that the MSC secretome includes, in addition to immunomodulators impacting on lung inflammation, additional factors with antiproliferative activity that may suppress vascular remodeling by their action on VSMCs. 7 Open in new tabDownload slide MSC-CM inhibits pulmonary artery smooth muscle cell proliferation. (A): Cultured PASMCs were serum-deprived for 48 hours followed by treatment with MSC-CM or MLF-CM in the presence of FBS and their proliferation rate was quantified relative to treatment with FBS alone. *, p < .001 compared with −FBS or +MSC-CM treatment. (B): Dose-response effect of MSC-CM on PASMC proliferation. Experiment was repeated twice in triplicate. *, p < .05 compared with non-MSC-treated controls. Data are presented as means ± SD. Statistical analysis as in Figure 1. Abbreviations: FBS, fetal bovine serum; MLF-CM, mouse lung fibroblast-conditioned media; MSC-CM, mesenchymal stem cell-conditioned media; PASMC, pulmonary artery smooth muscle cell. 7 Open in new tabDownload slide MSC-CM inhibits pulmonary artery smooth muscle cell proliferation. (A): Cultured PASMCs were serum-deprived for 48 hours followed by treatment with MSC-CM or MLF-CM in the presence of FBS and their proliferation rate was quantified relative to treatment with FBS alone. *, p < .001 compared with −FBS or +MSC-CM treatment. (B): Dose-response effect of MSC-CM on PASMC proliferation. Experiment was repeated twice in triplicate. *, p < .05 compared with non-MSC-treated controls. Data are presented as means ± SD. Statistical analysis as in Figure 1. Abbreviations: FBS, fetal bovine serum; MLF-CM, mouse lung fibroblast-conditioned media; MSC-CM, mesenchymal stem cell-conditioned media; PASMC, pulmonary artery smooth muscle cell. Discussion Pulmonary hypertension is a condition associated with a variety of pulmonary disorders, including alveolar hypoxia. A major therapeutic challenge in PAH is to reverse lung vascular remodeling and prevent RV failure, the latter being the final pathway leading to death in these patients. Hypoxia exposure in mice is associated with an early peak in lung inflammatory cell accumulation, coupled with marked induction of proinflammatory cytokines and chemokines that subsides within a week of hypoxic exposure [26]. However, circulating monocytes/fibrocytes recruited at that early time may contribute significantly to the structural remodeling and persistent vasoconstriction of the pulmonary circulation weeks later [37], even if the alveolar inflammatory profile subsequently adjusts to reflect adaptation to a chronic hypoxic state. We have previously reported that transgenic mice with constitutive, lung-specific overexpression of HO-1 do not manifest the early inflammatory peak and are protected from PAH on continued exposure to hypoxia [26]. It has not been obvious whether the protective function(s) of HO-1 reside solely with early immunomodulation or if HO-1 overexpression can reverse established disease once severe hypoxia-induced PAH has developed. In this study, we investigated both the prevention as well as reversal of PAH using WT and Hmox1KO animals exposed to chronic hypoxia. Mice were transplanted with MSCs from transgenic lines that have been engineered to overexpress the hHO-1 transgene in the lung epithelium under either a constitutive (SH01 line) or a conditional (CC77 line) promoter. It is relevant to note that in the CC77 line, the CCSP-promoter-driven rtTA is expressed in Clara cells and, ectopically, in type II cells [31]. Activation of the hHO-1 transgene on Dox administration follows the rtTA specificity of expression and therefore, in both systems, hHO-1 overexpression in the recipient lung requires phenotypic conversion of donor MSCs to lung epithelial cells. We have tested both SH01 and CC77 donor MSC cultures to verify that in vitro expression of hHO-1 was absent (results not shown). Therefore, the observed expression of hHO-1 in the recipient lung and augmentation of MSC efficacy must reside with the rare donor cells transdifferentiating into lung epithelium. Fusion of donor MSCs and recipient epithelial cells is apparently not the major mechanism of hHO-1 activation. Engraftment and transdifferentiation were further verified by examining Y-FISH fluorescein isothiocyanate (FITC) signals and using confocal techniques to ensure signal specificity. Accordingly, in WT mice, the combination of CC77-MSCs and Dox treatments prevented the development of PAH just as SH01-MSC treatment reversed disease in WT recipients. Only recipients treated with Dox were fully protected, whereas CC77-MSC treatment without Dox conferred partial protection from hypoxia-induced injury, similar to results obtained with WT-MSC transplantation. The validation of the results from both donor transgenic lines clearly indicates that, although MSCs possess innate mechanisms conferring protection against PAH, hHO-1 overexpression by rare cells engrafted in the lung epithelium augments their efficacy and restores RVSP to normal range. In this context, it is important to note that only SH01-MSC treatment was sufficient to eliminate the formation of mural thrombus that is a characteristic response of Hmox1KO exposed to chronic hypoxia. Similar to the low level of lung engraftment, rare MSCs were detected in the hearts of recipient animals 2 weeks postinjection (data not shown). As the hHO-1 transgene cannot be activated in the heart, the observed protection cannot be the result of cardiac HO-1 overexpression. This suggests that the cardiac protection observed in our system is the indirect result of lowered lung vascular resistance or due to paracrine factors secreted into the circulation by lung-resident donor cells [33], but the nature of mechanisms involved in cardiac protection merits further investigation. MSC engraftment in the lung occurs more rarely than certain original reports assessed, and paracrine functions are now recognized to be a major therapeutic component of MSC therapy in certain models of lung disease [15, 17, 19, 33]. In our model, we observed a rapid clearance of infused MSCs within 2 days post-transplantation, in line with reports estimating the level of engraftment to be less than 0.01% [38]. Our results also parallel studies showing that intratracheal MSC administration producing rare engraftment in pulmonary and cardiac tissues can attenuate monocrotaline-induced PAH and endothelial dysfunction and that transplantation of MSCs constitutively overexpressing eNOS can improve RV impairment and increase survival [12–14]. Although full reversal of hypoxia-induced PAH in WT mice was associated with the potential of MSCs to overexpress hHO-1 in the recipient lung on transdifferentiation, accurate assessment of relative hHO-1 mRNA levels at different time points after transplantation is challenging in the context of total lung RNA, as they are orders of magnitude less than endogenous mRNAs. Nevertheless, we observed an intriguing correlation between levels of hHO-1 mRNA and levels of Y chromosome by deriving an index of hHO-1 expression per resident donor cell. The peak of hHO-1 mRNA observed at 7 days postinjection suggests that a cohort of resident MSCs activated epithelial promoters at that time point, presumably subsequent to transdifferentiation into type II cells. We can only speculate that the decrease in hHO-1 mRNA per resident MSC after day 7 may be the result of further differentiation of most donor-derived type II cells into type I cells. More importantly, our data show that a transient peak in hHO-1 expression augments the efficacy of MSC treatment, preventing PAH development in WT mice, inhibiting RV thrombus in Hmox1KO mice, and reversing established disease in WT animals. This apparent paradox of a prominent physiologic effect in the absence of significant MSC engraftment or sustained detectable levels of hHO-1 expression may result from the potent immunomodulating properties of the MSC secretome as others have also postulated [15, 17, 33]. Accordingly, the bolus of MSC paracrine factors tilts the recipient's immune system balance toward an anti-inflammatory equilibrium that persists even in the presence of a continuing hypoxic insult. Indeed, using the BPD model, we have reported that a single injection of MSC-CM protects the recipient animals for up to 2 weeks of hyperoxia [19]. In this report, we suggest that the MSC bolus modulates the inflammatory response to hypoxia by decreasing the levels of proinflammatory cytokines, CCL2 (MCP-1) and IL-6, and increasing the level of anti-inflammatory IL-10. We and others have shown that these cytokines are regulated by hypoxia and participate in cascades leading to the development of lung vascular disease in various models of PAH [26, 39–41]. As hypoxia-induced lung inflammation peaks within the first week of hypoxia, a transient increase in lung HO-1 levels during this time, combined with the paracrine immunomodulatory action of MSCs, synergize to prevent the later development of PAH. We note that, unlike the bleomycin injury mouse model or the monocrotaline rat model of PAH, the acute hypoxia-induced proinflammatory cytokine release and cellular infiltrate have subsided after 5 weeks of hypoxia, the time we performed MSC transplantation in our PAH reversal model. Nevertheless, MSC therapy ameliorated lung vascular disease, and as in the prevention model, HO-1 overexpression augmented MSC efficacy, completely reversing disease in WT animals. These findings point to additional cytoprotective actions of MSCs and HO-1 on the lung vasculature that may be independent of their immunomodulating effects. We have previously reported [35] that HO-1 and its enzymatic product, CO, inhibit cell cycle progression, proliferation, and migration of smooth muscle cells and, as we show in this report, MSCs secrete factor(s) that strongly inhibit the proliferative effect of mitogens on VSMCs. Combined, the growth-inhibitory actions of HO-1-derived CO and the antiproliferative factors in the MSC secretome apparently act synergistically on VSMCs to reverse lung vessel wall remodeling and reduce PAH. It is also tempting to speculate that additional cytoprotective actions of MSCs include their ability to alter the pool size and reparative capacity of tissue-specific progenitor/stem cells. In this context, it is relevant to mention that studies on our hyperoxia-induced BPD model suggest that MSC treatment can increase both the overall number of bronchioalveolar stem cells (BASCs) and the number of junctions scoring positive for these progenitors [42]. Intriguingly, a characteristic of these progenitors is their ability to activate both the CCSP and SP-C promoters, therefore, both the hHO-1 transgenes we used in these studies should be expressed in BASCs. This supports the speculation that protective actions of MSCs and/or HO-1 on the injured lung go beyond immunomodulatory effects and include direct inhibition of smooth muscle cell growth, or potentially, augmentation of the function of cell types responsible for maintaining pulmonary homeostasis. Conclusion We used several model systems to investigate the effects of MSC transplantation on hypoxia-induced pulmonary hypertension and derive additional insights into potential mechanism(s) of MSC action. Results from multiple genetically modified animal lines that include constitutive and inducible lung epithelial cell-specific overexpression of the hHO-1 transgene demonstrate that a small fraction of donor cells can transdifferentiate into lung alveolar epithelium and significantly augment the efficacy of MSC treatment by overexpressing HO-1. Importantly, the likely paracrine actions of MSCs include the previously recognized immunomodulatory effects on lung inflammation and, as we report here, potent antiproliferative effects on pulmonary VSMC growth as well as potential cardioprotective effects. Exploiting the unique characteristics of MSCs as vehicles of gene delivery, modulators of hypoxia-induced inflammation, and inhibitors of VSMC growth may provide more effective approaches to not only preventing but also reversing diseases of the pulmonary and systemic vasculature. Disclosure of Potential Conflicts of Interest The authors indicate no potential conflicts of interest. 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Google Scholar OpenURL Placeholder Text WorldCat Author notes Author contributions: O.D.L.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; S.A.M.: conception and design, provision of study material or patients, collection and/or assembly of data, data analysis and interpretation, manuscript writing; M.S.C.: collection and/or assembly of data; E.V.: collection and/or assembly of data; C.L.: collection and/or assembly of data; M.A.: collection and/or assembly of data; A.F.-G.: collection and/or assembly of data, X.L.: provision of study material or patients; R.B.: provision of study material or patients; S.K.: conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript. First published online in STEM CELLS EXPRESS October 18, 2010. Disclosure of potential conflicts of interest is found at the end of this article. Telephone: 617-919-2355; Fax: 617-730-0260 Copyright © 2010 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Mesenchymal Stromal Cells Expressing Heme Oxygenase-1 Reverse Pulmonary Hypertension
JF - Stem Cells
DO - 10.1002/stem.548
DA - 2011-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/mesenchymal-stromal-cells-expressing-heme-oxygenase-1-reverse-CJsB5Q6uKG
SP - 99
EP - 107
VL - 29
IS - 1
DP - DeepDyve
ER -