Vasoprotective effect of PDGF-CC mediated by HMOX1 rescues retinal degeneration

Significance PDGF-CC plays critical roles in many biological processes, such as development, tumor growth, and angiogenesis. However, its role in blood vessel survival/regression and the underlying mechanisms remain unknown. Here, using different loss- and gain-of-function assays and multiple model systems, we show that PDGF-CC is a critical vascular protective factor required to maintain blood vessel survival. Mechanistically, we found that heme oxygenase-1 (HMOX1) activity is crucial for the vascular protective/survival effect of PDGF-CC. Given the general involvement of vascular degeneration in most degenerative diseases, PDGF-CC may be of therapeutic use in treating different types of degenerative disorders. Our findings point out that the PDGF-CC level should be monitored closely in various pathological conditions to ensure normal blood vessel survival. Blood vessel degeneration is critically involved in nearly all types of degenerative diseases. Therefore strategies to enhance blood vessel protection and survival are highly needed. In this study, using different animal models and cultured cells, we show that PDGF-CC is a potent vascular protective and survival factor. PDGF-CC deficiency by genetic deletion exacerbated blood vessel regression/degeneration in various animal models. Importantly, treatment with PDGF-CC protein not only increased the survival of retinal blood vessels in a model of oxygen-induced blood vessel regression but also markedly rescued retinal and blood vessel degeneration in a disease model of retinitis pigmentosa. Mechanistically, we revealed that heme oxygenase-1 (HMOX1) activity is critically required for the vascular protective/survival effect of PDGF-CC, because blockade of HMOX1 completely abolished the protective effect of PDGF-CC in vitro and in vivo. We further found that both PDGF receptors, PDGFR-β and PDGFR-α, are required for the vasoprotective effect of PDGF-CC. Thus our data show that PDGF-CC plays a pivotal role in maintaining blood vessel survival and may be of therapeutic value in treating various types of degenerative diseases.

B lood vessel degeneration and regression are vital pathologies in numerous human diseases and are associated with nearly all types of degenerative diseases, such as retinitis pigmentosa (RP), diabetic retinopathy, age-related macular degeneration, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (1)(2)(3). RP is a retinal degenerative disorder in which blood vessel degeneration contributes significantly to retinal atrophy, ultimately leading to loss of vision. In addition, recent studies have shown that prolonged treatment with antiangiogenic drugs may cause tissue degeneration (4)(5)(6). Given the increasing incidence of many degenerative diseases in an aging population and the rapidly growing clinical use of antiangiogenic drugs, there is an urgent need for strategies promoting blood vessel survival and protection. Because the pathological process of vascular degeneration involves complex mechanisms (7), treating such diseases remains challenging. Identifying effective vascular protective factors and the underlying mechanisms therefore is highly warranted.
However, whether PDGF-CC plays a role in the survival/regression of blood vessels remains unknown thus far.
In this study we used different animal models and cultured cells and investigated the potential effect of PDGF-CC on blood vessel survival. We found that PDGF-CC is a potent vasoprotective factor that rescues blood vessels from degeneration/regression under developmental and pathological conditions. Mechanistically, we show that heme oxygenase-1 (HMOX1), a potent antioxidative and anti-inflammatory factor, is required for the vasoprotective effect of PDGF-CC. Our data indicate that PDGF-CC may be of therapeutic use in treating different types of degenerative diseases in which blood vessel survival is impaired.
Results pdgf-c Deficiency Increases Blood Vessel Regression. Although PDGF-CC is highly expressed in the vascular system (8,19), its role in blood vessel survival is unclear. We therefore used pdgfc-deficient mice (Fig. S1A) and various model systems to investigate this role. First, a model of hyaloid blood vessel regression Significance PDGF-CC plays critical roles in many biological processes, such as development, tumor growth, and angiogenesis. However, its role in blood vessel survival/regression and the underlying mechanisms remain unknown. Here, using different loss-and gain-of-function assays and multiple model systems, we show that PDGF-CC is a critical vascular protective factor required to maintain blood vessel survival. Mechanistically, we found that heme oxygenase-1 (HMOX1) activity is crucial for the vascular protective/survival effect of PDGF-CC. Given the general involvement of vascular degeneration in most degenerative diseases, PDGF-CC may be of therapeutic use in treating different types of degenerative disorders. Our findings point out that the PDGF-CC level should be monitored closely in various pathological conditions to ensure normal blood vessel survival. showed lower hyaloid vessel densities at postnatal day 3 (P3) and P8 in pdgf-c-deficient mice (n = 8, P < 0.01, Fig. 1A; n = 8, P < 0.05, Fig. S1B), demonstrating that pdgf-c deficiency increases hyaloid vessel regression. It is noteworthy that no difference in hyaloid vessel density was observed at P1 (n = 8, P > 0.05) (Fig.  1A), suggesting the unlikelihood of a developmental defect. Secondly, a model of oxygen-induced retinal blood vessel regression (OIR) showed that pdgf-c deficiency led to larger avascular areas 5 d after hyperoxia (n = 8, P < 0.05) (Fig. 1B), demonstrating that pdgf-c deficiency accelerates oxygen-induced retinal blood vessel regression. Again, no difference in retinal blood vessel density was observed in neonatal pdgf-c-deficient mice (D n = 8, P > 0.05) (Fig. S1C), suggesting the unlikelihood of a developmental defect.

PDGF-CC Protects Retinal Blood Vessels from Oxygen-Induced
Regression. We subsequently investigated whether PDGF-CC could protect blood vessels from regression using an OIR model (21,22), which includes two stages to assess the degree of vessel regression. In the first stage (75% O 2 ), hyperoxia induced retinal blood vessel regression from P7 to P12 ( Fig. 2A, Fig. S2A, BSAtreated, and Fig. S2B, Upper Left). In the second stage (room air), severe retinal hypoxia caused by the preceding retinal vessel regression led to retinal neovascularization at P17 (Fig. 2B, Upper Left and Fig. S2A, BSA-treated). Therefore, if a potent vasoprotective factor was supplied in the first stage to prevent retinal blood vessels from regression in the first place, retinal neovascularization would not occur in the second stage, as indeed was the case for PDGF-CC. In the first stage of this model, under this specific experimental condition, intravitreal injection of PDGF-CC (500 ng per eye) decreased oxygen-induced retinal blood vessel regression by more than 50% at various time points (n = 8, P < 0.001) ( Fig. 2A and Fig. S2B), whereas PDGF-AA, another ligand of PDGFR-α, had little effect, and PDGF-BB and  inhibited blood vessel regression, leading to a smaller avascular area as measured by IB4 staining after 5 d of hyperoxia at P12. PDGF-BB and PDGF-DD had some effect, but PDGF-AA had no effect. (B) After 5 d of room air at P17, retinae treated with PDGF-CC displayed better retinal blood vessel regrowth with fewer avascular areas and less hypoxia-induced neovascularization than in the BSA control. Retinae treated with PDGF-BB and PDGF-DD had somewhat reduced avascular areas and hypoxia-induced neovascularization. Each image of a whole-mount retina shown in A and B represents a mosaic of several individual images. (C) Immunofluorescent staining using markers for ECs, pericytes, and SMCs showed that PDGF-CC treatment decreased vessel regression, leading to more CD31 + , NG2 + , and SMA + blood vessels at both P8 and P12. The ratios of the staining intensities of CD31/NG2 and CD31/SMA did not differ in the BSA-and PDGF-CC-treated samples. (Scale bars: 300 μm in A and B; 50 μm in C; 10 μm in C, Insets.) **P < 0.01, ***P < 0.001.
PDGF-DD had weaker effects (n = 8, P < 0.001 and P < 0.01, respectively) ( Fig. 2A and Fig. S2B). In the second stage of this model (after 5 d of room air), PDGF-CC-treated retinas displayed little hypoxia-induced retinal neovascularization and smaller avascular areas than the BSA-treated control (n = 8, P < 0.001 and P < 0.01, respectively) ( Fig. 2B), but in retinae treated with PDGF-BB and PDGF-DD, these two parameters were more similar to those in the BSA control. Immunoprecipitation assays and immunofluorescent staining showed that the PDGF proteins activated PDGFR-α and PDGFR-β in cultured vascular smooth muscle cells (SMCs) and endothelial cells (ECs), respectively (Fig. S3). In addition, PDGF-CC treatment led to more CD31 + , neural/glial antigen 2 (NG2) + , and smooth muscle actin (SMA) + vessels at both P8 and P12 ( Fig. 2C and Fig. S1E) but did not result in more blood vessels than what can be seen in normal retinae (n = 8, P < 0.001) (Fig. S2C). In addition, the ratios of the staining intensities of CD31/NG2 and CD31/SMA show no difference between BSA-and PDGF-CC-treated samples (Fig. 2C), indicating that PDGF-CC seems to affect all the vascular cells.
PDGF-CC Up-Regulates HMOX1 Expression in Vitro and in Vivo. To explore the mechanism underlying the vasoprotective/survival effect of PDGF-CC, we investigated the genes regulated by PDGF-CC. Real-time PCR showed that PDGF-CC treatment up-regulated the expression of HMOX1, a potent anti-oxidative gene (23), and other prosurvival genes in the retinae 24 h after hyperoxia ( Fig. 3A and Fig. S4A). Indeed, PDGF-CC inhibition or blocking PDGFR-α, the receptor for PDGF-CC, using neutralizing antibodies markedly decreased HMOX1 expression (Fig. 3 B and C) and inhibited PDGF-CC-induced PDGFR-α activation in the retinae (Fig. S4F). At a protein level, Western blot showed higher HMOX1 levels after PDGF-CC treatment in  different vascular cells, whereas pdgf-c deficiency decreased HMOX1 expression in the retinae (Fig. 3D). PDGF-AA, another ligand of PDGFR-α, did not up-regulate HMOX1 expression (Fig. S4E), and PDGF-BB and PDGF-DD had a weaker effect than PDGF-CC (Fig. S4 B and C). Thus, PDGF-CC is a potent inducer of HMOX1.
Vasoprotection by PDGF-CC via HMOX1 in Vitro and in Vivo. We next investigated the role of HMOX1 in PDGF-CC-mediated survival of vascular cells. Different vascular cells were cultured in serumfree medium so that their survival (rather than proliferation) could be investigated. We found that PDGF-CC increased the survival of both vascular ECs and SMCs (n = 6, P < 0.05, P < 0.01, P < 0.001) (Fig. 3 E-H). Importantly, cotreatment with an HMOX1 inhibitor, zinc protoporphyrin (ZnPP IX) (n = 6, P < 0.05, P < 0.01, P < 0.001) (Fig. 3 E-H), or HMOX1 siRNA (n = 6, P < 0.05) (Fig. 3 I and J and Fig. S5A) abolished the survival effect of PDGF-CC. In an OIR model in vivo, intravitreal injection of PDGF-CC protected retinal vessels from regression and markedly reduced avascular areas (n = 6, P < 0.001) (Fig. 4). Importantly, coadministration of the HMOX1 inhibitor ZnPP IX completely abolished the vasoprotective effect of PDGF-CC in vivo in a dose-dependent manner (n = 6, P < 0.001) (Fig. 4), demonstrating that the vascular protective/survival effect of PDGF-CC requires HMOX1.

PDGF-CC Rescues Blood Vessel and Retinal Degeneration via HMOX1
in an RP Model. We next tested whether PDGF-CC could ameliorate blood vessel degeneration under pathological conditions using rd1 mice, a commonly accepted model for retinitis pigmentosa, in which retinal and blood vessel degeneration represents a serious pathology by displaying few blood vessels and thin retinae (Fig. 5 A and C, BSA-treated). In this model, intravitreal injection of PDGF-CC protein rescued retinal blood vessels from degeneration and resulted in higher blood vessel densities throughout the retinae (n = 4, P < 0.001 or P <0.05) (Fig. 5A and Fig. S5B). The effect of PDGF-CC was particularly striking in the deep retinal layer where blood vessel density increased by about eight-fold (n = 4, P < 0.01) (Fig. 5B). Importantly, apart from the increased survival of retinal blood vessels, PDGF-CC treatment also rescued retinal degeneration, as shown by the increased thickness of different retinal layers (n = 4, P < 0.05, P < 0.01, P < 0.001) (Fig. 5C and Fig. S5C). It is noteworthy that coadministration of the HMOX1 inhibitor ZnPP IX abolished the effect of PDGF-CC nearly completely (n = 4, P < 0.05, P <0.01, P <0.001) (Fig. 5 A-C and Figs. S5 B  and C and S6 A and B). GFAP staining showed no significant difference in overall astrocyte activation/density between PDGF-CC-and BSA-treated retinae even though there seemed to be a tendency of increased GFAP staining in the nerve fiber layer (Fig. S4G). Thus, PDGF-CC rescued both retinal and blood vessel degeneration via HMOX1 under pathological conditions. PDGFR-α and PDGFR-β Are Required for the Vasoprotective Effect of PDGF-CC. PDGF-CC binds to and activates PDGFR-α and PDGFR-β (12,15). We therefore investigated their potential role in the vasoprotective effect of PDGF-CC. Immunofluorescent staining detected PDGFR-α and PDGFR-β expression on retinal blood vessels (Fig. 6A and Fig. S7). Immunoprecipitation and immunoblotting showed activation of PDGFR-α and PDGFR-β by PDGF-CC in vascular SMCs and ECs (Fig. 6B). PDGFR-α and PDGFR-β activation in the PDGF-CC-treated retinae also was detected by fluorescent staining in both the OIR and RP models in vivo (Fig. S8 A and B). Moreover, the survival/ protective effect of PDGF-CC was abolished by PDGFR-β and PDGFR-α neutralizing antibodies in cultured vascular SMCs and ECs (n = 5, P < 0.05, P < 0.01, P < 0.001) (Fig. 6 C-E). Furthermore, in the OIR model, the vasoprotective effect of PDGF-CC was largely abolished by neutralizing antibodies against PDGFR-α and PDGFR-β (n = 6, P < 0.001). The effect of blocking the PDGFR pathways was similar to pdgf-c deletion in pdgf-c knockout mice (n = 6, P < 0.001) (Fig. 6F), demonstrating the requirement of PDGFR-β and PDGFR-α for the survival/ protective effect of PDGF-CC on blood vessels.

Discussion
Blood vessel degeneration and regression have become an important focus in biomedical research because of their critical involvement in nearly all types of degenerative diseases. Therefore, there is an urgent need to identify effective molecules for vascular protection and survival. In this study we show that PDGF-CC is a potent vascular protective/survival factor that rescues retinal and blood vascular degeneration, whereas its deficiency exacerbates blood vessel regression in various animal models. Importantly, we revealed a previously unidentified mechanism underlying the function of PDGF-CC by showing that HMOX1 is critically required for the vascular survival effect of PDGF-CC.
Even though PDGF-CC has been shown to be a potent angiogenic factor (10,11,15,(18)(19)(20), its role in blood vessel survival and protection remained unclear. The receptors for PDGF-CC, PDGFR-α, and PDGFR-β, are thought to be expressed mainly by vascular SMCs, pericytes, and other mesenchymal cells and have been shown to be important for vascular survival because their inhibition led to vascular degeneration (24). However, it remained unknown whether PDGF-CC plays a role in this process and whether it has a direct effect on vascular ECs. In this study, we found expression and activation of PDGFR-β and PDGFR-α by PDGF-CC not only in vascular SMCs but also in ECs. Indeed, neutralizing antibodies against PDGFR-β and PDGFR-α abolished the survival effects of PDGF-CC nearly completely, demonstrating that a direct survival effect of PDGF-CC on vascular SMCs and ECs is a major factor in PDGFR-βand PDGFR-α-mediated blood vessel survival and protection.
The genes downstream of the PDGF-CC pathway are not well studied thus far. In this study, we found that PDGF-CC markedly up-regulated the expression of HMOX1 in different vascular cells, whereas inhibition of PDGF-CC or its receptors suppressed its expression in vitro and in vivo. Importantly, treatment with an HMOX1 inhibitor completely abolished the PDGF-CC-mediated vasoprotective effect on vascular SMCs and ECs in vitro and in various animal models in vivo. HMOX1 is a potent vasoprotective and survival factor because its robust antioxidative, antiapoptotic, and anti-inflammatory effects protect cells and tissues from injury (23). Given that oxidative stress, apoptosis, and inflammation are critically involved in most degenerative diseases, induction of HMOX1 expression has been considered a promising strategy for the treatment of degenerative diseases. As a potent HMOX1 inducer, PDGF-CC achieves its vasoprotective effect, at least partially, through HMOX1.
It is noteworthy that PDGF-CC protein treatment in the RP model of rd1 mice rescued not only blood vessel but also retinal degeneration, as shown by the improved retinal vessel density and retinal thickness. RP is a retinal degenerative disease characterized by the death of retinal photoreceptor cells and blood vessel degeneration (3). To mitigate retinal degeneration in RP effectively, both retinal blood vessels and photoreceptors must be protected. In this study, the rescue effect of PDGF-CC may have several aspects. First, PDGF-CC has a direct vasoprotective effect on retinal blood vessels, which subsequently provide better blood perfusion and thus better photoreceptor survival. Second, PDGF-CC also may have a direct neuroprotective effect on retinal photoreceptor cells, which express the PDGFRs (8,9). Indeed, we have shown previously that PDGF-CC is a potent neuronal survival factor (9). One potential issue that remains to be defined better in future studies is to investigate astrocyte activation in detail after PDGF-CC treatment, because there seems to be a tendency for increased GFAP staining in the nerve fiber layer in the PDGF-CC-treated retinae. Thus, PDGF-CC appears to be a potent survival factor that protects the retina from degeneration via multiple mechanisms.
In summary, using different loss-and gain-of-function assays and multiple model systems, we demonstrate here that PDGF-CC functions as a critical vascular protective factor that is required to maintain blood vessel survival. PDGFR-β and PDGFR-α mediate the vascular survival effect of PDGF-CC on multiple vascular cells by up-regulating HMOX1 expression. Given the general involvement of blood vessel defects in most degenerative disorders, PDGF-CC may be of therapeutic use in treating such diseases.

Materials and Methods
Models of Blood Vessel Regression/Degeneration. All animal experiments were approved by the Animal Use and Care Committee of Zhongshan Ophthalmic Center at the Sun Yat-Sen University, Guangzhou, People's Republic of China. pdgf-c-deficient mice, the OIR model, the hyaloid blood vessel regression model, and the RP model of rd1 mice are described in SI Materials and Methods.
Cell Culture, Survival Assay, and Immunofluorescence Staining. Detailed descriptions of cell culture, the survival assay, and immunofluorescence staining procedures can be found in SI Materials and Methods. Cell culture, survival assays, and immunofluorescence staining were performed as described in refs. 9, 19, 21, 22, and 25. All cell-culture experiments were performed in triplicate and were repeated twice.
Receptor Activation, Western Blot, and Real-Time PCR. Detailed descriptions of receptor activation, Western blot, and real-time PCR procedures can be found in SI Materials and Methods and Table S1. Receptor activation, Western blot, and real-time PCR were performed as described in refs. 9, 19, and 22.
Statistics. Two-tailed Student t test and one-way ANOVA were used for statistical analysis. Differences were considered statistically significant when P < 0.05. All values are presented as mean ± SEM of the number of determinations.