Initiation and maintenance of angiogenesis in angiogenic diseases is a complex process requiring modulation of numerous pro- and anti-angiogenic molecules operating through complex intracellular signaling pathways. Identification of the key instigators of this process will help in defining future therapies for controlling vascularization. Here, for the first time to our knowledge, we have isolated micro-regions of angiogenic and quiescent microvessels from brain tissue of patients who died from acute ischaemic stroke and compared expression of the key angiogenic genes using real-time PCR and TaqMan microfluidity cards. From nanogram quantities of material, we have identified up-regulation of 7 genes with key roles in promotion of angiogenesis. Immunohistochemistry demonstrated a specific association of Tie-2, MCP-1, MMP-2 and HGF-α in peri-infarcted and infarcted CD105-positive blood vessels.
Laser-capture microdissection and RNA amplification technology has allowed the possibility to isolate and examine specific micro-sized cellular areas from heterogeneous tissue components. Previously, Hashimoto et al
], isolated vascular rich areas from synovial tissues and performed single-real-time PCR analysis on individual genes following RNA extraction. They demonstrated up-regulation of VEGF/VEGFR, HIF-1α, and inhibitor of differentiation-2 in blood vessels from inflamed regions of patients with rheumatoid arthritis. LCM was used to compare blood vessels from glioblastoma multiforme with those from vessels in normal brain tissue [14
]. Pre-amplification of RNA followed by microarray analysis showed up-regulation of genes including insulin-like growth factor binding protein-7 and SPARC. Roy et al
], compared global gene expression between blood vessels isolated by laser-capture from normal skin and identified de-regulated genes from those within chronic wounded tissue, utilizing Ulex Europaeus Agglutinin (UEA1) which binds specifically to EC, to highlight the vessels. We also found that rapid staining with UEA1 in sterile water did not degrade the RNA whilst rapid immunostaining employing buffers did (our unpublished data). To discriminate between active and quiescent vessels, we labeled serial sections staining in groups of four using serial reference staining with anti-CD105, anti-CD31 and anti-Flt-1 antibodies for vessel identification. We confirmed that the areas chosen contained enriched markers of EC activation using RNA pre-amplification technology and house keeping controls. This showed that the relative amplification of genes was similar. One of the main aims of this work was to identify expression of angiogenic and anti-angiogenic factors produced in micro-regions of brain tissue in association with active micro-vessels. Therefore, in these experiments we carefully dissected concentrated areas of vessels including closely associated ECM encompassing any inflammatory components. In this way, we were able to gain an insight into the microenvironment to which the growing vessels were existing. Expression or synthesis of genes directly by the endothelial cells would feature as the main constituent due to their high relative concentration (identified by histology in all samples), however any component consisting of secreted factors from inflammatory infiltrates would also be seen giving us an overall view of the composition of these micro-hotspots.
Real-time PCR analysis of our microfluidity card data showed significant correlation in expression between de-regulated angiogenic genes and our markers of EC activation (CD105 and Flt-1) confirming the validity of our methodology. MCP-1 was originally identified as an important chemokine responsible for activation of macrophages and monocytes during inflammation but now is known to have a direct effect on EC mitogenesis in vitro and vessel formation in vivo [16
]. The molecular mechanisms have not been dissected although Niu et al
, demonstrated up-regulation of MCP-1-induced protein was necessary for VEGF and HIF-1α induction in HUVEC. We have shown that MCP-1 is strongly associated with active microvessels in peri-infarcted regions undergoing tissue remodeling after stroke.
We also showed a significant increase in Tie-2 expression in stroked regions. Both angiopoietin 1 and 2 can bind to the tyrosine kinase receptor Tie-2, which is responsible for vessel maturation and stability including facilitation of smooth muscle cell/pericyte attachment and therefore could be a key promoter of revascularization after stroke [18
]. Simvastatin, used in treatment to lower cholesterol, is also angiogenic, and studies have shown that treatment with this drug following MCAO in a rat model, significantly increased EC capillary tube-formation dependent on induction of Tie-2 [19
]. Studies using animal models have suggested that treatment with bone marrow stromal cells (MSC) after stroke, increases angiogenesis and tissue reperfusion in association with increased Tie-2 expression [20
]. The same authors showed that capillary-like structure formation in mouse brain EC was increased in the presence of supernatant derived from MSC, whilst knock-down of Tie-2 inhibited this, suggesting an important role for Tie-2 in revascularization.
Hemorrhagic incident occurring after cerebral ischemia may be related to damage of the microvascular basal lamina of the brain, and can aggravate cerebral ischemia. This may be associated with up-regulation of MMPs and in particular, MMP-2 [21
]. MMP-2 is up-regulated in EC exposed to inflammatory cytokines such as interleukin-1-beta and growth factors including nerve growth factor, where in vivo
, it promotes capillary invasion and so is probably increased in active stroke regions undergoing remodelling [22
]. Dong et al
], showed that resveratrol treatment 24 h–7 days after MCAO in mice increased MMP-2 and VEGF expression and concomitantly, the number of cortical microvessels as well as the neurological score, suggesting that MMP-2 has an important role in modulation of angiogenesis after stroke. This is in agreement with our data showing its association with CD105-positive microvessels in peri-infarcted regions.
We showed that expression of HGF-α was increased in the small neo-tubular vessels from peri-infarcted regions. Injection of human HGF gene with a hemagglutinising virus into rat CSF after MCAO, reduced neurological deficit within 24 hours of treatment and increased the number of microvessels in stroke-affected tissue [25
]. The same authors showed that HGF-α gene transfer could significantly improve recovery of learning and memory concomitant with increased angiogenesis and neurite extension after stroke [26
]. Rush et al
], demonstrated that addition of HGF-α to human brain microvessel EC, stimulated their migration through signalling pathways involving JNK, ERK and c-Src. This, together with the fact that HGF/c-met is also a chemoattractant for stroke-mobilized bone-marrow-derived stem cells [28
], indicates that HGF could be a prime target for angiogenic therapy after stroke. Here, we also showed that HBMEC exposed to OGD demonstrated up-regulated HIF-1α and Hsp70 concomitant with MCP-1/MMP-2 and Tie-2 gene and protein expression suggesting at least some of the proteins may be produced by EC de novo after stroke.
For correct angiogenesis and maturation of vessels to take place, the time of expression and the number and type of angiogenic molecules effective in the vicinity of the developing microvessels, may be important, and this may vary dependent on the surrounding matrix. In our other studies (submitted elsewhere), we have shown using the same technology and identical TaqMan microarrays that the hypoxic environment associated with neovesel activation in carotid neointimal plaques induces expression of an overlapping, but certainly not identical group of angiogenic factors. In this case Tie-2 was also over-expressed as seen in this study, whilst the receptor for advanced glycation end-products (RAGE), angiopoietin-1 and Notch-3 were only increased in the plaque vascular bed. Therefore, the rate at which new microvessels are formed and/or are able to mature is probably governed by the number and concentration of relevant factors expressed. This may have important consequences in relation to attempts to induce therapeutic angiogenesis for the production of mature intimal vessels less prone to leakage and rupture, and the process may be site-specific. Future studies should aim to examine the effects of modulating these factors in terms of ratio and concentration with a view to optimising stable re-vascularization in vivo.
One of the ultimate clinical goals is to enhance and modulate the body's response to collateral blood vessel formation to maximise brain tissue reperfusion as rapidly as possible after stroke. Although we have not studied all the identified proteins in detail, pilot studies showed that β-catenin (pro-angiogenic and also mobilizes endothelial progenitor cells; [29
] and TIMP-1 were also expressed strongly in stroke-affected microvessels, whilst c-Kit-positive cells were also present (data not shown).
Some of the limitations of this study include the small numbers of measured samples which has not allowed us to relate the findings directly to clinical information and stroke characteristics such as infarct size, extent of recovery, association with time of survival and survival. More extended detailed studies using larger patient cohorts could examine the information gained from this study and ascertain the importance of these proteins in mediating tissue reperfusion and any relationship with improvement in patient survival. Similarly, the data we have provided only allows us to infer what the overall effects of the presence of mixtures of both pro- and anti-angiogenic factors on microvessel formation, proliferation and maturation after stroke might be. Future experiments employing matrigel implant models may be able to determine the effects of introducing a mixture of factors such as those described in this study and examining in detail the formation structure and maturation of vessels over time.