BACKGROUND
stroke is a leading cause of death and disability in the western world.
neuronal survival in peri-infarcted regions determines the extent of patient recovery  <cit> .
patients with a higher density of blood vessels have reduced morbidity and mortality  <cit> .
restoration of cerebral microvascular circulation following angiogenesis/revascularization in peri-infarcted regions may salvage tissue, enhance neuronal survival and enhance functional recovery after stroke  <cit> .
following rat middle cerebral artery occlusion , new blood vessels initiated through vascular buds, formed regular connections with intact microvessels within one week of ischaemia, with patterns similar to those in normal brain  <cit> .
in disease situations, abnormally behaving cells are surrounded by heterogeneous tissue elements, and the areas of interest/diseased cells may constitute less than 5% of the volume of a sample.
conventional technology have employed microarrays to identify general changes in gene and protein regulation in biopsies from normal and abnormal regions of atherosclerotic plaques  <cit>  and stroke tissue  <cit> , but have failed to discover cell-specific changes, and in particular, those associated with angiogenesis.
we hypothesise that within active regions of remodelling, angiogenic and non-angiogenic areas may co-exist.
laser-capture microdissection  can be used to isolate microvessels in evolving lesions.
when combined with the latest rna microscale extraction and analysis technology, this provides a powerful and sensitive tool for identification of genetic changes associated with blood vessel activation.
cd <dig> is the best known marker of active endothelial cells  in diseased angiogenic tissues and is of prognostic value and a potential target for anti-angiogenic therapy in a variety of solid tumours  <cit> .
cd <dig> is expressed by active ec making it the perfect target for identification of regions of tissue remodelling after stroke.
the expression of cd <dig> in brain after stroke has not been studied, however, cd <dig> expression was induced by hypoxia in murine brain microvascular ec via mitogen activated protein kinase  pathways  <cit>  suggesting it is also a marker of active neovessel formation in ischaemic tissues.
using cd <dig> and flt- <dig> as discriminating markers of microvessel activation, we aimed to identify the molecular fingerprint responsible for neovessel activation and revascularization following stroke.
RESULTS
areas rich in cd105-positive or cd31-positive/cd105-negative vessels were chosen for laser-capture as shown in .
the cdna obtained from  <dig> ng of total rna was pre-amplified using the taqman applied biosystems preamp master mix kit .
pre-amplification of rna demonstrated equality of multiplication of key genes
pre-amplification analysis demonstrated that flt- <dig>  cd <dig> and cd <dig> amplified to a similar extent and within acceptable limits with ΔΔct lower than the cut off point of  <dig>  and no significant differences between the cts of any of the post-amplified genes .
equality of gene amplification was tested using control , and cd <dig>  cd <dig> and flt- <dig>
selected immuno-positive cd105-positive tissue samples were enriched with cd <dig> and flt- <dig> gene expression
confirmation was made that the samples were cd31/cd105/flt- <dig> enriched using taqman real-time pcr.
micro-areas chosen by laser-capture on the basis of cd105/flt- <dig> ihc demonstrated significant increase in their gene expression  and a strong correlation was also seen between cd105/gus and flt-1/gus in all samples indicating accurate sampling of angiogenic and non-angiogenic regions .
no significant difference in expression of cd <dig> in paired samples was seen .
taqman microfluidity card comparison of samples revealed significant differences in expression of key angiogenic genes correlating with cd <dig> and flt- <dig> gene expression
of the  <dig> genes tested,  <dig> pro-angiogenic genes were significantly up-regulated in cd105/flt- <dig> positive vessel containing regions  and a further one with rq increased to > than  <dig>  in 4/ <dig> samples .
none of the anti-angiogenic genes present in the arrays was significantly modified.
immunohistochemistry confirmed expression of hgf-α, mcp <dig>  mmp- <dig> and tie- <dig> in microvessels from stroked brain regions
hgf-α was expressed strongly in microvessels of varying size from all active stroke regions after stroke .
old infarcted regions with dead cells were not stained.
in contrast, there was no observable expression of hgf-α in normal looking blood vessels from the contralateral region.
glia and neurons were-unstained however, occasional inflammatory cells were positively stained in stroked regions.
many of the hgf-α-positive vessels, particularly those with malformed/impatent morphology stained cd <dig> positive suggesting cellular activation or angiogenesis was occurring in these areas .
tie- <dig> showed a similar pattern with strong staining in small and medium sized, blood vessels from peri-infarcted and infarcted regions .
again, there with no observable staining in sections from normal looking contralateral tissue .
d; tie- <dig> also co-localised in cd105-positive immature neo-vessels suggesting an association with angiogenesis.
tie- <dig> expression was specific for blood vessels with no staining of glia or inflammatory cells; however occasional dying neurons from the infarcted core had weak cytoplamic staining.
mmp- <dig> was strongly expressed in both relative mature and immature microvessels from stroked regions, particularly in penumbral regions undergoing active remodeling  and also in cells with the morphological appearance of astrocytes  and neurons .
normal looking .
mcp- <dig> stained primarily cd105-positive microvessels in peri-infarcted regions with some positive vessels also in the infarcted zones .
normal looking tissue did not stain for mcp- <dig> .
some glia and infiltrating inflammatory cells were positively stained for mcp- <dig>  in all cases, old infarcted regions stained negatively for all the antibodies tested and the majority of staining was seen in active regions of remodeling and revascularization.
a summary of the findings is given in table  <dig>
exposure of hbmec to ogd in vitro resulted in up regulation of tie- <dig>  mcp- <dig> and mmp- <dig> protein
when semi-confluent hbmec were exposed to ogd for  <dig> h, increased gene expression of tie- <dig> , mcp- <dig>  and mmp- <dig>  were obtained by real-time pcr.
values were controlled using the house keeping gene gus and a concomitant increase in expression of hif-1α  and hsp <dig>  demonstrated a strong response to the hypoxic environment .
all experiments were repeated twice and a representative example is shown.
immuno-staining showed that an increase in protein intensity was seen in the cytoplasm of pi-positive hbmec following ogd  .
discussion
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  <dig> genes with key roles in promotion of angiogenesis.
immunohistochemistry demonstrated a specific association of tie- <dig>  mcp- <dig>  mmp- <dig> 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,  <cit> , 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- <dig> 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  <cit> .
pre-amplification of rna followed by microarray analysis showed up-regulation of genes including insulin-like growth factor binding protein- <dig> and sparc.
roy et al,  <cit> , 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  which binds specifically to ec, to highlight the vessels.
we also found that rapid staining with uea <dig> in sterile water did not degrade the rna whilst rapid immunostaining employing buffers did .
to discriminate between active and quiescent vessels, we labeled serial sections staining in groups of four using serial reference staining with anti-cd <dig>  anti-cd <dig> and anti-flt- <dig> 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 , 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  confirming the validity of our methodology.
mcp- <dig> 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  <cit> .
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- <dig> is strongly associated with active microvessels in peri-infarcted regions undergoing tissue remodeling after stroke.
we also showed a significant increase in tie- <dig> expression in stroked regions.
both angiopoietin  <dig> and  <dig> can bind to the tyrosine kinase receptor tie- <dig>  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  <cit> .
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- <dig>  <cit> .
studies using animal models have suggested that treatment with bone marrow stromal cells  after stroke, increases angiogenesis and tissue reperfusion in association with increased tie- <dig> expression  <cit> .
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- <dig> inhibited this, suggesting an important role for tie- <dig> 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- <dig>  <cit> .
mmp- <dig> 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  <cit> .
dong et al,  <cit> , showed that resveratrol treatment  <dig> h– <dig> days after mcao in mice increased mmp- <dig> and vegf expression and concomitantly, the number of cortical microvessels as well as the neurological score, suggesting that mmp- <dig> 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  <dig> hours of treatment and increased the number of microvessels in stroke-affected tissue  <cit> .
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  <cit> .
rush et al,  <cit> , 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  <cit> , 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 hsp <dig> concomitant with mcp-1/mmp- <dig> and tie- <dig> 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 , 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- <dig> was also over-expressed as seen in this study, whilst the receptor for advanced glycation end-products , angiopoietin- <dig> and notch- <dig> 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 .
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.
CONCLUSIONS
we hypothesise that combined processes including matrix degradation, direct activation of migration and proliferation of endothelial cells, attraction of bone marrow-derived stem cells and stabilization of new vessels with pericytes and smooth muscle cells are all vital components of this response, which is maintained many weeks after stroke in active regions of remodelling and therefore future therapeutic treatments might involve maximisation of all of these processes.

 stroke is a leading cause of death and disability in the western world. neuronal survival in peri-infarcted regions determines the extent of patient recovery. patients with a higher density of blood vessels have reduced morbidity and mortality. restoration of cerebral microvascular circulation following angiogenesis/revascularization in peri-infarcted regions may salvage tissue, enhance neuronal survival and enhance functional recovery after stroke. following rat middle cerebral artery occlusion, new blood vessels initiated through vascular buds, formed regular connections with intact microvessels within one week of ischaemia, with patterns similar to those in normal brain. in disease situations, abnormally behaving cells are surrounded by heterogeneous tissue elements, and the areas of interest/diseased cells may constitute less than 5% of the volume of a sample. conventional technology have employed microarrays to identify general changes in gene and protein regulation in biopsies from normal and abnormal regions of atherosclerotic plaques and stroke tissue, but have failed to discover cell-specific changes, and in particular, those associated with angiogenesis. we hypothesise that within active regions of remodelling, angiogenic and non-angiogenic areas may co-exist. laser-capture microdissection can be used to isolate microvessels in evolving lesions. when combined with the latest rna microscale extraction and analysis technology, this provides a powerful and sensitive tool for identification of genetic changes associated with blood vessel activation. cd is the best known marker of active endothelial cells in diseased angiogenic tissues and is of prognostic value and a potential target for anti-angiogenic therapy in a variety of solid tumours. cd is expressed by active ec making it the perfect target for identification of regions of tissue remodelling after stroke. the expression of cd in brain after stroke has not been studied, however, cd expression was induced by hypoxia in murine brain microvascular ec via mitogen activated protein kinase pathways suggesting it is also a marker of active neovessel formation in ischaemic tissues. using cd and flt- as discriminating markers of microvessel activation, we aimed to identify the molecular fingerprint responsible for neovessel activation and revascularization following stroke.  areas rich in cd105-positive or cd31-positive/cd105-negative vessels were chosen for laser-capture as shown in. the cdna obtained from ng of total rna was pre-amplified using the taqman applied biosystems preamp master mix kit. pre-amplification of rna demonstrated equality of multiplication of key genes pre-amplification analysis demonstrated that flt- cd and cd amplified to a similar extent and within acceptable limits with ΔΔct lower than the cut off point of and no significant differences between the cts of any of the post-amplified genes. equality of gene amplification was tested using control, and cd cd and flt- selected immuno-positive cd105-positive tissue samples were enriched with cd and flt- gene expression confirmation was made that the samples were cd31/cd105/flt- enriched using taqman real-time pcr. micro-areas chosen by laser-capture on the basis of cd105/flt- ihc demonstrated significant increase in their gene expression and a strong correlation was also seen between cd105/gus and flt-1/gus in all samples indicating accurate sampling of angiogenic and non-angiogenic regions. no significant difference in expression of cd in paired samples was seen. taqman microfluidity card comparison of samples revealed significant differences in expression of key angiogenic genes correlating with cd and flt- gene expression of the genes tested, pro-angiogenic genes were significantly up-regulated in cd105/flt- positive vessel containing regions and a further one with rq increased to > than in 4/ samples. none of the anti-angiogenic genes present in the arrays was significantly modified. immunohistochemistry confirmed expression of hgf-α, mcp mmp- and tie- in microvessels from stroked brain regions hgf-α was expressed strongly in microvessels of varying size from all active stroke regions after stroke. old infarcted regions with dead cells were not stained. in contrast, there was no observable expression of hgf-α in normal looking blood vessels from the contralateral region. glia and neurons were-unstained however, occasional inflammatory cells were positively stained in stroked regions. many of the hgf-α-positive vessels, particularly those with malformed/impatent morphology stained cd positive suggesting cellular activation or angiogenesis was occurring in these areas. tie- showed a similar pattern with strong staining in small and medium sized, blood vessels from peri-infarcted and infarcted regions. again, there with no observable staining in sections from normal looking contralateral tissue. d; tie- also co-localised in cd105-positive immature neo-vessels suggesting an association with angiogenesis. tie- expression was specific for blood vessels with no staining of glia or inflammatory cells; however occasional dying neurons from the infarcted core had weak cytoplamic staining. mmp- was strongly expressed in both relative mature and immature microvessels from stroked regions, particularly in penumbral regions undergoing active remodeling and also in cells with the morphological appearance of astrocytes and neurons. normal looking. mcp- stained primarily cd105-positive microvessels in peri-infarcted regions with some positive vessels also in the infarcted zones. normal looking tissue did not stain for mcp-. some glia and infiltrating inflammatory cells were positively stained for mcp- in all cases, old infarcted regions stained negatively for all the antibodies tested and the majority of staining was seen in active regions of remodeling and revascularization. a summary of the findings is given in table exposure of hbmec to ogd in vitro resulted in up regulation of tie- mcp- and mmp- protein when semi-confluent hbmec were exposed to ogd for h, increased gene expression of tie-, mcp- and mmp- were obtained by real-time pcr. values were controlled using the house keeping gene gus and a concomitant increase in expression of hif-1α and hsp demonstrated a strong response to the hypoxic environment. all experiments were repeated twice and a representative example is shown. immuno-staining showed that an increase in protein intensity was seen in the cytoplasm of pi-positive hbmec following ogd. discussion 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 genes with key roles in promotion of angiogenesis. immunohistochemistry demonstrated a specific association of tie- mcp- mmp- 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- 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. pre-amplification of rna followed by microarray analysis showed up-regulation of genes including insulin-like growth factor binding protein- 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 which binds specifically to ec, to highlight the vessels. we also found that rapid staining with uea in sterile water did not degrade the rna whilst rapid immunostaining employing buffers did. to discriminate between active and quiescent vessels, we labeled serial sections staining in groups of four using serial reference staining with anti-cd anti-cd and anti-flt- 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, 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 confirming the validity of our methodology. mcp- 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. 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- is strongly associated with active microvessels in peri-infarcted regions undergoing tissue remodeling after stroke. we also showed a significant increase in tie- expression in stroked regions. both angiopoietin and can bind to the tyrosine kinase receptor tie- 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. 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-. studies using animal models have suggested that treatment with bone marrow stromal cells after stroke, increases angiogenesis and tissue reperfusion in association with increased tie- expression. 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- inhibited this, suggesting an important role for tie- 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-. mmp- 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. dong et al,, showed that resveratrol treatment h– days after mcao in mice increased mmp- and vegf expression and concomitantly, the number of cortical microvessels as well as the neurological score, suggesting that mmp- 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 hours of treatment and increased the number of microvessels in stroke-affected tissue. 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. 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, 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 hsp concomitant with mcp-1/mmp- and tie- 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, 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- was also over-expressed as seen in this study, whilst the receptor for advanced glycation end-products, angiopoietin- and notch- 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. 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.  we hypothesise that combined processes including matrix degradation, direct activation of migration and proliferation of endothelial cells, attraction of bone marrow-derived stem cells and stabilization of new vessels with pericytes and smooth muscle cells are all vital components of this response, which is maintained many weeks after stroke in active regions of remodelling and therefore future therapeutic treatments might involve maximisation of all of these processes. 
areas were compared for pro- and anti-angiogenic gene expression using targeted taqman microfluidity cards containing genes and real-time pcr. angiogenesis is critical for the development of new microvessels and leads to re-formation of collateral circulation, reperfusion, enhanced neuronal survival and improved recovery. angiogenesis correlates with patient survival following acute ischaemic stroke, and survival of neurons is greatest in tissue undergoing angiogenesis. here, we have isolated active and inactive micro-vessel rich-regions from stroke-affected and contralateral tissue of patients using laser-capture micro-dissection.
 angiogenesis correlates with patient survival following acute ischaemic stroke, and survival of neurons is greatest in tissue undergoing angiogenesis. angiogenesis is critical for the development of new microvessels and leads to re-formation of collateral circulation, reperfusion, enhanced neuronal survival and improved recovery.  here, we have isolated active and inactive micro-vessel rich-regions from stroke-affected and contralateral tissue of patients using laser-capture micro-dissection. areas were compared for pro- and anti-angiogenic gene expression using targeted taqman microfluidity cards containing genes and real-time pcr. further analysis of key gene de-regulation was performed by immunohistochemistry to define localization and expression patterns of identified markers and de novo synthesis by human brain microvessel endothelial cells was examined following oxygen-glucose deprivation. our data revealed that seven pro-angiogenic genes were notably up-regulated in cd positive microvessel rich regions. these were, beta-catenin, neural cell adhesion molecule, matrix metalloproteinase-, tissue inhibitor of matrix metalloproteinase-, hepatocyte growth factor-alpha, monocyte chemottractant protein- and and tie- as well as c-kit. immunohistochemistry demonstrated strong staining of mmp- hgf-alpha, mcp- and tie- in stroke-associated regions of active remodeling in association with cd positive staining. in vitro, ogd stimulated production of tie- mcp- and mmp- in hbmec, demonstrated a de novo response to hypoxia.  in this work we have identified concurrent activation of key angiogenic molecules associated with endothelial cell migration, differentiation and tube-formation, vessel stabilization and stem cell homing mechanisms in areas of revascularization. therapeutic stimulation of these processes in all areas of damaged tissue might improve morbidity and mortality from stroke. 