In this study, VPA markedly reduced infarct volume and improved functional recovery on day 14 after MCAO in rats. Concurrently, VPA treatment enhanced post-ischemic angiogenesis by increasing microvessel density, facilitating endothelial cell proliferation, and upregulating rCBF in the ipsilateral cortex. In addition to enhancing HDAC inhibition, VPA potentiated MCAO-induced HIF-1α accumulation and upregulated VEGF protein levels and MMP-2/9 activities in the ipsilateral cortex on days 7 and 14 post-MCAO. Furthermore, HIF-1α inhibition reversed the enhanced post-ischemic angiogenesis and functional recovery observed after VPA treatment. Our findings suggest that (1) chronic VPA treatment enhances post-ischemic angiogenesis and promotes long-term functional recovery in an experimental model of ischemic stroke, and (2) the pro-angiogenic effects of VPA likely involve HDAC inhibition and upregulation of HIF-1α and pro-angiogenic factors VEGF and MMP-2/9.
Accumulating evidence has established that angiogenesis naturally occurs after brain ischemia in humans and animals, potentially functioning as an endogenous mechanism to restore oxygen and nutrient supply to affected brain tissue.4
However, post-ischemic angiogenesis is often insufficient to improve clinical outcomes. Our results demonstrated that despite higher microvessel density, rCBF was significantly lower in the ipsilateral versus contralateral hemisphere on day 14 after MCAO. This finding echoes a clinical study demonstrating that the infarct region of post-mortem stroke patients contains a higher proportion of empty microvessels than normal brain tissue.1
Additionally, a rat MCAO study indicated that ischemia induces a transient population of leaky microvessels.20
Taken together, these observations suggest that the newly-formed microvessels may not be perfused or functional. In the current study, VPA treatment strongly potentiated both microvessel density and rCBF in the ischemic hemisphere, suggesting that VPA may facilitate microvessel formation as well as perfusion. Furthermore, it has been proposed that ischemia-triggered angiogenesis requires additional factors, such as brain-derived neurotrophic factor (BDNF), for long-term stabilization.21
VPA can induce BDNF in rat cortical neurons by activating BDNF promoter IV through HDAC inhibition,22
suggesting that VPA may also stabilize post-ischemic angiogenesis by inducing BDNF expression.
VEGF expression rapidly increases within hours and remains elevated for weeks after ischemia in rodent and human brains.6–7
Late administration of exogenous VEGF in the ischemic penumbra of rats enhances angiogenesis and improves neurological recovery.23
MMP-9 activity is elevated 2–4 days after stroke in the post-mortem human brain, whereas MMP-2 activity is elevated at later intervals.5
Delayed MMP inhibition in the peri-infarct cortex suppresses neurovascular remodeling and functional recovery on day 14 in ischemic rats.8
Consistent with these studies, we found that VEGF protein levels and MMP-2/9 activities were significantly increased on days 7 and 14 after MCAO. VPA treatment markedly enhanced the upregulation of VEGF and MMP-2/9, suggesting that VEGF and MMP-2/9 contribute to VPA’s ability to enhance post-ischemic angiogenesis.
Both VEGF and MMPs increase BBB permeability in the acute phase and subsequently facilitate neurovascular remodeling after stroke.11, 23
In the present study, VPA enhanced post-ischemic angiogenesis by upregulating VEGF protein expression and MMP-2/9 activities on day 14 after MCAO. We recently demonstrated that VPA attenuates BBB disruption and brain edema by suppressing MMP-9 induction and tight junction degradation 24 hours after MCAO.16
In a swine hemorrhagic shock model, VPA mitigates pathologic endothelial cell function by attenuating the overexpression of VEGF and its receptor 6 hours after resuscitation.24
Therefore, VPA appears to have a dual role in preserving post-ischemic endothelial cell function: it limits cell damage by inhibiting MMP-9 and VEGF in the early phase, whereas it enhances angiogenesis by upregulating VEGF and MMP-2/9 in the later recovery phase.
HIF-1 regulates pro-anigogenic genes after hypoxia/ischemia, and its activation is predominantly controlled by α subunit stabilization.12
In this study, VPA treatment significantly enhanced MCAO-induced HIF-1α protein accumulation in the ipsilateral cortex on days 7 and 14 after MCAO. 2ME2, a HIF-1α inhibitor, completely abolished the ability of VPA to increase microvessel density and improve rotarod performance. These findings suggest that the pro-angiogenic effects of VPA involve regulation of HIF-1α and are critical for the ability of VPA to improve long-term functional recovery after ischemia. It is suggested that VEGF and MMP-2/9 are under transcriptional regulation of HIF-1.12
In support of this notion, we found that HIF-1α inhibition suppressed VEGF and MMP-2/9 upregulation induced by VPA treatment in MCAO rats. To confirm that HIF-1α can directly upregulate VEGF and MMP-2/9, HIF-1α was induced in primary rat brain microvascular endothelial cells (RBMVECs), and VEGF and MMP-2/9 mRNA levels were measured by quantitative real-time PCR. Cobalt chloride (CoCl2
) is widely used to mimic hypoxia by stabilizing HIF-1α.25–26
treatment increased HIF-1α protein levels with a peak observed at 6 hours (Supplemental Figure S5A
). At this time point there was also a significant increase in VEGF and MMP-2/9 mRNA levels in RBMVECs (Supplemental Figure S5C
). Pretreatment with 2ME2 significantly suppressed HIF-1α upregulation (Supplemental Figure S5B
). Consequently, 2ME2 almost completely inhibited CoCl2
-induced VEGF mRNA increase, partially reduced the MMP-9 mRNA increase, but did not affect MMP-2 mRNA levels (Supplemental Figure S5C
). Together with the in vivo
data, these findings indicate that upregulation of HIF-1α results in the elevation of VEGF and MMP-2/9, although it is possible that HIF-1α differentially regulates the transcription of these three genes.
The precise mechanisms underlying VPA-enhanced HIF-1α accumulation and angiogenesis in ischemic brain remain unclear. VPA is a pan-inhibitor of HDAC class I (1, 2, 3, 8 isoforms) and IIa (4, 5, 7, 9 isoforms).14
VPA-induced HDAC inhibition results in histone hyperacetylation, chromatin relaxation and gene transcription. As an index of HDAC inhibition, acetylation of histone-H3 and H4 was robustly increased by VPA treatment, especially on day 14 after MCAO, suggesting that HDAC inhibition may participate in the pro-angiogenic effects of VPA following ischemia. In support of this notion, a recent in vitro
study shows that HDAC inhibitors, VPA and suberoylanilide hydroxamic acid (SAHA), greatly enhance VEGF-induced spheroid sprout formation in endothelial cells, and that VPA displays a trend toward increasing endothelial cell migration.27
Additionally, VPA potentiates extracellular signal-regulated kinase 1/2 activation in endothelial cells, which is known to promote cell survival and angiogenesis.27
Furthermore, HDAC inhibition can induce the differentiation of multipotent adult progenitor cells into endothelial cells, with or without VEGF co-stimulation.28
Besides endothelial cells, microvascular pericytes also play an important role in optimizing post-insult angiogensis. In human microvascular pericytes, a qPCR angiogenesis array showed that VPA leads to a general increase in genes associated with vessel stabilization and maturation, such as endothelial survival, endothelial tube formation/stabilization/branching, and maintenance of direct cell-cell contacts between endothelial cells and pericytes.29
Taken together, these findings suggest that VPA-induced HDAC inhibition may modulate pro-angiogenic gene expression and contribute to post-ischemic angiogenesis.
Interestingly, HDAC inhibitors are currently undergoing clinical evaluation as potential anti-cancer therapies because of their anti-angiogenic effects in tumors.30
VPA has been shown to inhibit HIF-1α stabilization and tumor angiogenesis in diverse cancer cell lines.31–32
However, there is no evidence showing the effects of VPA on HIF-1α and angiogenesis in noncancerous brain cells, especially after ischemia. In line with the in vivo
findings, in an in vitro
oxygen-glucose deprivation (OGD) model, we found that 3 hours OGD and 24 hours reperfusion increased HIF-1α protein levels in RBMVECs, and this increase was significantly augmented by treatment with 1 mmol/L VPA (Supplemental Figure S6A
). In addition, VPA also significantly increased cell viability after OGD insult (Supplemental Figure S6B
). VPA alone did not affect HIF-1α protein levels or cell viability under normoxic conditions. These findings provide complementary evidence supporting the pro-angiogenic effects of VPA after brain ischemia, conditions that likely differ significantly from those in cancer models.
Notably, existing studies suggest the likelihood that each HDAC isoform interacts differently with angiogenic pathways under specific conditions. There are also controversial findings regarding specific HDAC isoforms associated with HIF-1α in different cancer cell lines. For instance, one study found that HDAC7 directly interacts with HIF-1α and increases its transcriptional activity in HEK293 cells.33
Another study in C2 cells showed that VPA inhibits HIF-1α only at high concentrations that are effective against class II HDACs, and further demonstrated that HDAC4 and 6, instead of HDAC7, are involved.32
In contrast, protein kinase D-dependent phosphorylation and nuclear export of HDAC5 and 7 mediates VEGF-induced angiogenesis in endothelial cells.34–35
Additionally, HDAC5 silencing has been shown to increase endothelial cell migration, sprouting, and tube formation, whereas HDAC5 overexpression decreases sprout formation.36
An electron microscopy study has demonstrated that the pattern of new blood vessels in the ischemic brain is similar to that in normal brain, but differs from that in growing tumors.37
Therefore, it is conceivable that interactions between HDAC isoforms and HIF-1α may be dissimilar in endothelial cells and cancer cells under different oxygen tension conditions, and that the pan-inhibition of HDACs by VPA would affect the post-ischemic angiogenesis differently than it would affect angiogenesis in cancer.
To our knowledge, this study is the first to demonstrate that VPA enhances post-ischemic angiogenesis in vivo. This may contribute to its observed effects in improving long-term functional outcome after ischemic stroke. Our findings lead to a better understanding of the beneficial effects of VPA against ischemic stroke, and pave the way for potential clinical trials.