Loss Of Methyl-CpG Binding Domain Protein 2 (MBD2) Enhances Endothelial Angiogenesis And Protects Mice Against Hindlimb Ischemic Injury

1. IntroductionEndothelial cells (ECs) line vessels in all organs/tissues and regulate the transport of nutrient substances, diverse biologically active molecules and blood cells. They act as the key and initial defense of vascular diseases and play a wide variety of critical roles in the control of vascular function. Therefore, endothelial dysfunction is the leading cause for cardiovascular diseases. For example, endothelial dysfunction is an essential factor for diabetes-induced vascular diseases which are the principal causes of death and disability in diabetic patients. Because the etiology for vascular diseases involves both genetic and epigenetic factors, it is a formidable challenge to identify an optimal therapeutic target with minimal side effect.As a major epigenetic component, altered patterns of DNA methylation are commonly seen in animals and patients with vascular diseases. Recent studies suggest that methylation of DNA can act as a "footprint" reflecting the consequence of environmental exposures on different types of cells. This pattern of DNA methylation can be "read" by a conserved family of methyl-CpG binding domain (MBD) proteins which includes MBD1, MBD2, MBD3, MBD4 and MeCP2. They associate with protein partners to play active roles in DNA methylation-mediated transcriptional repression and/or heterochromatin formation. As a result, they are responsible for creating, maintaining, and interacting with the epigenome. Each of the MBD proteins is distinguished by its binding affinity to the methylated DNA, and the highest binding affinity was noticed for MBD2, but mammalian MBD3 fails to selectively recognize methylated DNA. Animals deficient for MeCP2, MBD1, MBD2 and MBD4 are viable except for MBD3 which leads to embryonic lethality. Mice lacking MeCP2 or MBD1 are associated with specific neurological defects, while MBD4 is found to suppress CpG mutability and tumorigenesis. In contrast, mice deficient for MBD2 show an almost normal phenotype except for minor abnormal maternal behavior observed in female mice, suggesting that MBD2 could be a favorable target for epigenetic therapy.In the present study, we sought to examine the role of MBD2 in endothelial function. We hypothesize that MBD2, as a key epigenetic regulator, modulates endothelial-restricted gene expression, and implicates in the regulation of angiogenesis and endothelial dysfunction. We found that suppression of MBD2 expression significantly enhanced angiogenesis and prevented Ecs from H2O2-induced apoptosis. Mice deficient for MBD2 showed a significant improved perfusion recovery after hindlimb ischemic injury. Remarkably, these mice were completely protected from diabetes-induced endothelial dysfunction. Given the fact that MBD2 has virtually no discernable effects on animals in the physiological condition, our results suggest that MBD2 could be a unique target for prevention and reversal of endothelial dysfunction in disease condition.2. Knockdown of MBD2 Promotes Angiogenesis and Protects Ecs from H2O2-induced ApoptosisWe first used human umbilical vein endothelial cells (HUVECs, ATCC) transfected with either an MBD2 or a control siRNA (Santa Cruz, CA) to examine the potential influence of MBD2 on EC function. Western blotting detected moderate levels of MBD2 in HUVECs, and the MBD2 siRNA dose-dependently suppressed MBD2 expression. MBD2 was almost undetectable when HUVECs transfected with 100nmol/L siRNA. We next seeded siRNA transfected HUVECs into culture plates preconditioned with growth factor-reduced Martigel (BD Bioscience, CA). Knockdown of MBD2 expression in HUVECs led to a significant enhanced capacity for tube formation. Typical tube formation was observed as early as 4h after seeding for MBD2 siRNA transfected cells, while almost no discernable tube formation was noticed for control siRNA transfected cells (data not shown). After 24h transfection the average total tube length (10.4±0.8mm) in each randomly selected field was significantly higher in MBD2 siRNA transfected cells than that of control siRNA transfected cells (3.3±0.5mm).To examine the impact of MBD2 on HUVEC proliferation, the transfected cells were cultured in the presence of 3H labeled thymidine followed by assay of incorporated radioactivity. Similarly, knockdown of MBD2 by siRNA significantly enhanced HUVEC proliferation as compared to the control cells. To demonstrate the effect of MBD2 on cndothelial apoptosis, HUVECs after 24h siRNA transfection were treated with 0.2mM H2O2 for 24h to induce apoptosis. Interestingly, knockdown of MBD2 by siRNA significantly protected HUVECs from H2O2-induced apoptosis. Together, these results suggest that MBD2 negatively regulates angiogenesis and promotes H2O2-induced endothelial apoptosis.3. Mice Deficient for MBD2 Have a Significant Improved Perfusion Recovery after Hindlimb IschemiaIn patients with cardiovascular disease, an attenuated angiogenic response to ischemia is associated with poor clinical outcomes. Given the observation that MBD2 negatively regulates endothelial angiogenesis, we examined its impact on perfusion recovery after ischemic injury. We first confirmed the absence of MBD2 protein in blood vessels of Mbd2+ mice. We next examined MBD2 temporal expression changes in wild-type (WT) mice undergoing hindlimb ischemia by a femoral artery excision. In the physiological condition, only very low levels of MBD2 were detected in the hindlimb. However, a steady increase for MBD2 was noticed upon ischemic induction, and the highest level was observed after day 4 of ischemic surgery. After which, MBD2 underwent a steady decrease and then returned to a relative low level after day 14 of surgery. Immunostaining revealed that MBD2 expression (in green) in the non-ischemic muscle sections was limited to the location of blood vessels, and was mainly localized to Ecs (CD31-positive, in red). In line with vascular neogenesis in response to ischemic insult, significant more Ecs (CD31-positive cells) were found along with much higher levels of MBD2 expression in the ischemic sections, suggesting that Ecs undergoing vascular neogenesis are associated with enhanced MBD2 expression.To determine the role of MBD2 in perfusion recovery, unilateral hindlimb ischemia was induced in Mbd2-/- and WT male mice. All mice had survived after surgical procedures. Blood flow in ischemic (left side) and nonischemic (right side) limbs was then monitored using a laser Doppler imaging system. Blood flow in the ischemic hindlimbs was almost undetectable in both Mbd2-/- and WT mice right after the femoral artery excision. Remarkably, Mbd2-/- mice showed partial blood flow restoration after day 2 of ischemic induction, and blood flow restored around 50% at day 7 and almost completely recovered at day 14 of ischemic insult. However, no visible blood flow restoration observed in WT mice until day 4 of ischemic induction, and blood flow only restored around 30% and 60% after days 7 and 14 of ischemic induction, respectively.To demonstrate the differences for neovascularization, we examined capillary (CD31-positive cells) and arteriole (smooth muscleα-actin positive cells) formation. It was consistently found that the number of CD31-positive cells (Ecs) andα-actin positive cells (smooth muscle cells) in the sections originated from ischemic Mbd2-/- mice were significantly higher than that of sections derived from WT ischemic mice. Taken together, our results demonstrate that loss of MBD2 promoted both angiogenesis and arteriogenesis associated with significant more effective response to hindlimb ischemia which then improved perfusion recovery after ischemic injury.4. Suppression of MBD2 Expression Activates EC Survival and Proangiogenic SignalsWe next examined two major EC survival signals, the extracellular signal-regulated kinase 1/2 (ERK1/2) and the serine-threonine kinase Akt, in siRNA transfected HUVECs. Interestingly, no significant difference was detected for Akt between MBD2 and control siRNA transfected HUVECs. However, much higher phosphorylated ERK1/2 (p-ERK1/2) was noticed in MBD2 siRNA transfected HUVECs, although total ERK1/2 did not change. Given the role of pERK1/2 in BCL-2 stabilization, we also noticed a significant higher levels of BCL-2 in MBD2 siRNA transfected HUVECs after H2O2 treatment. Furthermore, studies in blood vessels derived from diabetic Mbd2+ mice and their control counterparts showed similar trend for both ERK1/2 and BCL-2. Surprisingly, knockdown of MBD2 did not show a significant impact on MnSOD and Cu/ZnSOD expression, the two major enzymes responsible for detoxification of reactive oxygen species (ROS). Our results suggest that MBD2 regulates endothelial apoptosis probably by modulating the survival signals relevant to ERK1/2 and BCL-2 signaling.To study the mechanism underlying MBD2 regulation of angiogenesis, we analyzed eNOS and VEGF-R2 activity, the two key proangiogenesis signals2,3. Remarkably, knockdown of MBD2 increased both total and activated eNOS (p-eNOS), and much higher VEGF-R2 was also detected in MBD2 siRNA transfected HUVECs. Importantly, studies in blood vessels isolated from Mbd2+ mice and their control counterparts showed similar results. We further examined the activity of p38 mitogen-activated protein kinase (MAPK). MBD2 did not show a significant impact on total p38 but much higher activated p38 was observed in MBD2 siRNA transfected HUVECs, and studies in Mbd2+ blood vessels showed similar trend. Together, loss of MBD2 probably activates cell survival and proangiogenic signals to enhance EC survival and angiogenesis. 5. eNOS Synergizes with VEGF-R2 to Promote Endothelial Survival and AngiogenesisTo demonstrate the connection of above characterized signals, we treated HUVECs with VEGF in the presence of blockades either for VEGF-R2 or eNOS. VEGF stimulation did not affect total VEGF-R2, eNOS and ERK1/2 expression, but promoted VEGF-R2, eNOS and ERK1/2 activation along with enhanced BCl-2 expression. As expected, VEGF-R2 neutralizing antibody or inhibitor (SU1498) suppressed VEGF-induced VEGF-R2 activation and diminished VEGF-induced ERK1/2 activation and BCL-2 upregulation. Blockade of VEGF-R2 also led to a modest decrease for p-eNOS. Notably, blockade of eNOS by N (G)-nitro-L-arginine methyl ester (L-NAME), not only inhibited eNOS activation (p-eNOS) but also suppressed VEGF-induced p-VEGF-R2 along with a significant decrease for p-ERK1/2 and BCL-2 expression. These results suggest that ERK1/2 and BCL-2 are downstream molecules of eNOS and VEGF-R2 signaling. Our data also suggest a crosstalk between eNOS and VEGF-R2 signaling, in which eNOS plays a predominant role by synergizing with VEGF-R2 to enhance endothelial survival and to promote angiogenesis.To confirm the above conclusion, we next treated Mbd2+ and control mice after hindlimb ischemic surgery with an eNOS inhibitor, L-NAME. Administration of L-NAME significantly impaired blood flow restoration in both Mbd2+ and control mice. More significantly, L-NAME treatment completely abolished the protective effect seen in Mbd2+ mice as manifested by the similar extent of impaired perfusion after ischemic injury.6. MBD2 Binds to the High GC-containing Regions in the eNOS and VEGF-R2 PromoterPrevious studies suggested evidence supporting a role for DNA methylation regulating eNOS and VEGF-R2 transcription. We therefore hypothesized that MBD2 represses eNOS and VEGF-R2 expression by directly binding to the methylated CpG-elements in their promoter. Although no typical CpG island exists in the eNOS promoter, a region containing CpG-elements is found in the 5'-flanking region. On the contrary, bioinformatic analysis characterized two putative CpG islands in the VEGF-R2 promoter. To test our hypothesis, we first treated HUVECs with 5-aza-2'-deoxycytidine (5-azadC), a DNA methylation inhibitor. Remarkably, knockdown of MBD2 by siRNA has no perceptible effect on 5-azadC treated HUVECs, indicating that the effect of MBD2 involves DNA methylation. Chromatin immunoprecipitation (ChIP) was then employed to pull-down the MBD2/DNA complexes. Primers flanking the entire eNOS promoter/5'-flanking region and the putative CpG islands of VEGF-R2 were used to amplify the MBD2-targeted DNA using the resultant precipitates. No amplification was observed for all primers in the eNOS promoter, but primers (F9/R9 and F10/R10) in the 5'-flanking region yielded positive results. Surprisingly, among the three primer pairs used to flank the putative CpG islands for VEGF-R2, only primers (F3/R3) covering the region between-64 to+166 yielded positive results.To confirm the above ChIP assay results, we specifically examined the methylation states of 13 CpG-elements in the eNOS 5'-flaking region by bisulfite DNA sequencing. Indeed, we detected CpG methylation both in physiological and ischemic condition. We also characterized a significant DNA methylation turnover upon ischemic insult (hypoxia plus serum starvation) in HUVECs as evidenced by a one-fold increase for the methylation rate of these CpG-elements. EMSA was then carried out to further confirm that MBD2 binds to the methylated CpG-elements within this region. Biotin-labeled PCR products flanking+31 to+197 were first methylated by SssI methylase and then used as the probe to detect MBD2 binding activity. It was found that MBD2 bound to those methylated PCR products with high affinity. To address that MBD2 represses eNOS transcription by binding to those methylated CpG-elements, eNOS promoter (-1700 to+350) was subcloned into a pGL-2 vector (pGL-eNOS). A mutated eNOS promoter reporter (pGL-eNOSm), in which cytosines in all CpG-elements between-200 to+300 were mutated to adenosine, was also constructed. Luciferase assays were then performed using those in vitro methylated reporters along with a pcDNA-MBD2 plasmid in HUVECs, respectively. Consistently, only those methylated reporters showed differences for luciferase activities, in which loss of CpG-elements resulted in a one-fold increase for the reporter activities. In contrast, both pGL-eNOS and pGL-eNOSm showed similar reporter activities in unmethylated condition. Taken together, these results suggest that MBD2 represses eNOS and VEGF-R2 transcription by directly binding to the methylated CpG-elements in their promoter region7. MBD2 Repression of eNOS Transcription Involves Chromatin Remodeling and Is Confined to EcsMBD2-mediated transcriptional repression has been suggested to be associated with chromatin remodeling that involves HDACs. To check whether MBD2 repressing eNOS transcription also involves chromatin remodeling, we added trichostatin A (TSA), an HDAC inhibitor that suppresses eNOS transcription, into MBD2 or control siRNA transfected HUVECs. Surprisingly, the effect of MBD2 siRNA on eNOS expression was completely abolished by TSA treatment as manifested by that MBD2 or control siRNA transfected HUVECs after TSA treatment showed same levels of eNOS expression.TSA treatment has been shown to induce eNOS expression in non-endothelial cell types, we therefore wondered whether loss of MBD2 would lead to eNOS expression in non-endothelial cells. HeLa cells after siRNA transfection were then treated with either TSA or DMSO (control vehicle). Surprisingly, knockdown of MBD2 failed to induce eNOS expression in HeLa cells, but eNOS was detected in the same cells after TSA treatment. Studies in HEK293 cells obtained similar results (data not shown). Next, we analyzed eNOS expression in splenocytes isolated from Mbd2+ and control mice. eNOS was absent in both Mbd2+ and WT splenocytes. However, eNOS was induced in all cells following TSA treatment. Together, our results indicate that MBD2 repressing eNOS expression involves chromatin remodeling and its effect is confined to Ecs. 8. Conclusion1. We demonstrated strong evidence for MBD2 in the blood recover after hindlimb ischemia.2. Knockdown of MBD2 Promotes Angiogenesis and Protects Ecs from H2O2-induced Apoptosis3. Suppression of MBD2 Expression Activates EC Survival and Proangiogenic Signals.4. MBD2 Binds to the High GC-containing Regions in the eNOS and VEGF-R2 Promoter5. MBD2 Repression of eNOS Transcription Involves Chromatin Remodeling and Is Confined to Ecs.In summary, MBD2 binding to the 5'UTR of eNOS and VEGF-R2 promoter region will decrease the transcription of eNOS and VEGF-R2 and then depress endothelial function and angiogenesis. Compared to control mice, MBD2 knock out mice will protect mice from diabetes induced endothelial dysfunction and enhance blood recover after hindlimb ischemia.