Borja Fernández

Bone Marrow-Derived Cells Do Not Incorporate Into the Adult Growing Vasculature

Abstract— Bone marrow-Derived cells have been proposed to form new vessels or at least incorporate into growing vessels in adult organisms under certain physiological and pathological conditions. We investigated whether bone marrow-Derived cells incorporate into vessels using mouse models of hindlimb ischemia (arteriogenesis and angiogenesis) and tumor growth. C57BL/6 wild-type mice were lethally irradiated and transplanted with bone marrow cells from littermates expressing enhanced green fluorescent protein (GFP). At least 6 weeks after bone marrow transplantation, the animals underwent unilateral femoral artery occlusions with or without pretreatment with vascular endothelial growth factor or were subcutaneously implanted with methylcholanthrene-induced fibrosarcoma (BFS-1) cells. Seven and 21 days after surgery, proximal hindlimb muscles with growing collateral arteries and ischemic gastrocnemius muscles as well as grown tumors and various organs were excised for histological analysis. We failed to colocalize GFP signals with endothelial or smooth muscle cell markers. Occasionally, the use of high-power laser scanning confocal microscopy uncovered false-positive results because of overlap of different fluorescent signals from adjacent cells. Nevertheless, we observed accumulations of GFP-positive cells around growing collateral arteries (3-fold increase versus nonoccluded side, P <0.001) and in ischemic distal hindlimbs. These cells were identified as fibroblasts, pericytes, and primarily leukocytes that stained positive for several growth factors and chemokines. Our findings suggest that in the adult organism, bone marrow-Derived cells do not promote vascular growth by incorporating into vessel walls but may function as supporting cells.

VEGFR-1–Selective VEGF Homologue PlGF Is Arteriogenic: Evidence for a Monocyte-Mediated Mechanism

Abstract— Two signaling receptors for vascular endothelial growth factor (VEGF) in the vasculature are known with not yet well-understood roles in collateral vessel growth (arteriogenesis). In this study, we examined the involvement of the two VEGF receptors in arteriogenesis. Therefore, we used the VEGF homologue placenta growth factor (PlGF), which only binds to VEGFR-1 and VEGF-E, which only recognizes VEGFR-2. These peptides were locally infused over 7 days after ligation of the femoral artery in the rabbit. Evaluation of collateral growth by determining collateral conductance and angiographic scores demonstrated that the VEGFR-1–specific PlGF contributed significantly more to arteriogenesis than the VEGFR-2 specific VEGF-E. The combination of VEGF-E and PlGF did not exceed the effect of PlGF alone, indicating that cooperation of the two VEGF receptors in endothelial cell signaling is not required for arteriogenesis. In an in vitro model of angiogenesis, VEGF and VEGF-E were comparably active, whereas PlGF displayed no activity when given alone and did not further increase the effects of VEGF or VEGF-E. However, PlGF was as potent as VEGF when monocyte activation was assessed by monitoring integrin surface expression. In addition, accumulation of activated monocytes/macrophages in the periphery of collateral vessels in PlGF-treated animals was observed. Furthermore, in monocyte-depleted animals, the ability of PlGF to enhance collateral growth in the rabbit model and to rescue impaired arteriogenesis in PlGF gene–deficient mice was abrogated. Together, these data indicate that the arteriogenic activity observed with the VEGFR-1–specific PlGF is caused by its monocyte-activating properties.

Involvement of the Fibroblast Growth Factor System in Adaptive and Chemokine-Induced Arteriogenesis

Abstract— Fibroblast growth factors (FGFs) have been applied in a variety of therapeutic and experimental studies to improve collateral blood flow. However, the pathophysiological role and the temporospatial expression of the FGFs and their receptors during arteriogenesis have never been elucidated in vivo. Here, we report that collateral artery growth in its early phase is associated with an increased expression of FGF receptor-1 (FGFR-1) and syndecan-4 on mRNA and protein levels as well as with an increased kinase activity of FGFR-1 in a rabbit model of arteriogenesis. However, the mRNA levels of FGF-1 and -2 remained constant. Our data suggest that these growth factors are supplied by endothelial attracted monocytes that, in turn, produce and deliver the FGFs to growing collateral arteries. Monocyte chemoattractant protein-1-stimulated arteriogenesis was strongly reduced in rabbits by application of the FGF inhibitor polyanetholesulfonic acid, indicating that the monocyte-related arteriogenesis (as well as the unstimulated adaptation proper) is promoted by FGFs. In summary, this study shows that arteriogenesis is associated with an increased expression of the FGFRs at the site of the vessel, whereas the growth-promoting ligands are supplied by monocytes in a paracrine way.

Receptor-independent role of the urokinase-type plasminogen activator during arteriogenesis.

To define the role of the plasminogen activators (PAs) urokinase PA (uPA) and tissue PA (tPA) as well as the uPA receptor (uPAR) in arteriogenesis, we investigated their impact in a rabbit and mouse model of adaptive collateral artery growth. Collateral artery growth was induced by occlusion of the femoral artery in rabbit and wild‐type (WT) mice and in mice with targeted inactivation of uPA (uPA−/−), tPA (tPA−/−), or uPAR (uPAR−/−). Northern blot results revealed a significant up‐regulation of uPA but not uPAR or tPA in the early phase of arteriogenesis in rabbit and WT mice. This up‐regulation on RNA level was followed by an increased protein level and enzymatic activity. Impaired perfusion recovery upon femoral artery ligation was observed by laser Doppler analysis in vivo in uPA‐deficient mice but not in uPAR or tPA deficiency compared with WT mice. Immunohistochemical studies revealed an association of leukocyte infiltration with arteriogenesis in WT mice that was strongly reduced in uPA−/− but not in uPAR‐ or tPA‐deficient mice. We conclude that arteriogenesis is promoted by an uPA‐mediated infiltration of leukocytes that is not dependent on uPAR.

Cardiac overexpression of monocyte chemoattractant protein-1 in transgenic mice mimics ischemic preconditioning through SAPK/JNK1/2 activation

OBJECTIVE AND METHODSnAlthough a beneficial association between innate immunity and ischemic preconditioning has recently been proposed, the mechanisms responsible for this link are poorly understood. To test the hypothesis that pro-inflammatory cytokines have a beneficial role in the activation of the cell survival pathway mediated by ischemic preconditioning, we have studied transgenic mice with cardiac myocyte specific overexpression of murine monocyte chemoattractant protein-1 (MCP-1). The resistance to ischemia was studied by performing 45-min (with or without injection of the SAPK/JNKs inhibitor D-JNKI1) and 3-day left coronary artery occlusions as well as 45-min left coronary artery occlusion followed by 3 days of reperfusion. In addition, quantitative Western blot analyses for TNF-alpha, and SAPK/JNK1/2, ERK1/2 and p38 activity were performed.nnnRESULTSnInfarct size, expressed in percent of either the risk area or the left ventricle, was reduced in transgenic mice when compared with control after both, 45-min (14.7+/-2.6% vs. 52.0+/-2.4%; P<0.05) and 45-min occlusion followed by 3 days of reperfusion (23.2+/-1.8% vs. 30.0+/-1.8%; P<0.05) but it was not significantly different for 3-day occlusion. Western blot analyses showed significantly increased levels of TNF-alpha (1.8-fold) and phosphorylated-SAPK/JNK1/2 (1.5-fold) in transgenic hearts. Phosphorylated-ERK1/2, and phosphorylated-p38 levels were unchanged. Immunohistochemistry revealed that in transgenic mice monocytes/macrophages, lymphocytes, and fibroblasts are the source of TNF-alpha, whereas myocytes have increased phosphorylated-SAPK/JNK1/2 levels. In addition, injection of the SAPK/JNKs inhibitor D-JNKI1 partially abrogated the cardioprotective effect observed in untreated transgenic mice.nnnCONCLUSIONnOverexpression of MCP-1 by cardiomyocytes causes chronic infiltration and activation of leukocytes, resulting in elevated TNF-alpha secretion and SAPK/JNK1/2 activation. The activation of this pathway is in part responsible for the preconditioning effect of MCP-1 overexpression. These results show a possible beneficial link between innate immunity and ischemic preconditioning through MAP-kinase activation.

Angiogenesis-independent cardioprotection in FGF-1 transgenic mice

OBJECTIVEnThis study was performed to evaluate the cardioprotective role of acidic fibroblast growth factor-1 (FGF-1) in transgenic mice with cardiac-specific overexpression of human FGF-1.nnnMETHODSnMice were subjected to coronary artery occlusion for 15-75 min with a continuously recorded 3-lead electrocardiogram (ECG). Infarct size was measured and ERK-1 and -2 activity was assessed by Western blot analysis. Creatine kinase and lactate dehydrogenase activity as marker for cell viability were measured in isolated ventricular myocytes subjected to simulated ischemia.nnnRESULTSnInfarct development was markedly delayed in transgenics with first signs of myocardial infarction visible at 45 min after coronary artery occlusion compared to 15 min in wildtype. Maximal infarct size (60% of risk area) did not differ, but transgenics reached maximal infarction after 75 min compared to 45 min in wildtype animals. ECG revealed delayed Q-wave development and delayed ST-segment elevation in transgenics. Creatine kinase and lactate dehydrogenase release was significantly attenuated from isolated transgenic myocytes at 4 and 8 h after simulated ischemia. The delay in infarct development is partially due to a constitutive higher expression of the extracellular signal-regulated kinases ERK-1 and -2 in the myocardium of transgenics. Additionally, injection of the ERK-1/2 inhibitor UO126 decreased the cardioprotective effect of FGF-1.nnnCONCLUSIONnCardiac specific overexpression of FGF-1 provides cardioprotection at the level of the cardiac myocyte, independent from angiogenesis, and at least partially mediated via activation of the mitogen activated protein kinase (MAP) ERK-1 and -2.

Arteriogenesis is associated with an induction of the cardiac ankyrin repeat protein (carp)

OBJECTIVEnCollateral artery growth (arteriogenesis) can be induced in rabbit and mice by occlusion of the femoral artery. We aimed to identify genes that are differentially expressed during arteriogenesis.nnnMETHODSn24 h after femoral ligation or sham operation collateral arteries were isolated from New Zealand white rabbits, mRNAs were extracted and amplified using the SMART technique. cDNAs were subjected to suppression subtractive hybridization. The differential expression was confirmed by Northern blot, Real time PCR and Western blot. Additionally, the gene expression was modulated in vivo by application of cytokines via osmotic minipumps.nnnRESULTSnWe found the cardiac ankyrin repeat protein (carp) mRNA to be upregulated at 24 h and already at 6 h and 12 h after surgery as shown by Northern blot hybridization and real time PCR. The carp mRNA was also increased in our mouse model of arteriogenesis. Western blot results on nuclear extracts of rabbit collaterals 24 h after surgery indicated that carp, which we showed to be expressed in endothelial cells and smooth muscle cells of collateral arteries by immunohistochemistry, was also upregulated on the protein level. We infused MCP-1, TGF-beta1 or doxorubicin for 24 h in rabbits and found that only TGF-beta1 led to an additional increase of carp mRNA. Overexpression of carp in cos-1 cells resulted in a 3.7-fold increase of the immediate early gene egr-1.nnnCONCLUSIONSnOur results implicate that carp is associated with the initiation and regulation of arteriogenesis.

Osteoglycin expression and localization in rabbit tissues and atherosclerotic plaques

The localization of osteoglycin (OG), one of the corneal keratan sulfate proteoglycans, was studied in different normal rabbit tissues, as well as in atherosclerotic lesions, by means of in situ hybridization and immunohistochemistry. OG was associated with the vasculature of all the organs analyzed. Normal aortas showed abundance of the protein in the adventitia and focally in the media. Peripheral vessels showed OG localized only in the adventitia. OG mRNA was restricted to vascular smooth muscle cells, pericytes, and fibroblasts in aorta and skeletal muscle. In striated muscle, OG was abundant and distributed in foci around muscles and vessels, whereas in visceral muscle, the protein was homogeneously distributed throughout the extracellular matrix. In all the other organs studied, OG was only associated with the vasculature, with the exception of the lung and liver. In these two organs, the protein accumulated also around cartilage, alveoli, and hepatic duct. In atherosclerotic lesions, OG mRNA was down-regulated in the media and up-regulated in the activated endothelium and thick neo-intima, whereas the protein accumulated in the front edge of migrating smooth muscle cells. We conclude that OG is a basic component of the vascular extracellular matrix. OG also plays a role in atherosclerosis, and might be useful for therapeutic interventions. In addition, the possible involvement of OG in maintaining physical properties of tissues is discussed.

The ankyrin repeat containing SOCS box protein 5: a novel protein associated with arteriogenesis.

Arteriogenesis, the growth of pre-existing collateral arteries, can be induced in rabbit by occlusion of the femoral artery. In order to identify and characterize genes differentially expressed during the early phase of arteriogenesis, cDNA of collateral arteries 24h after femoral ligation or sham operation was subjected to suppression subtractive hybridization. We identified the ankyrin repeat containing SOCS box protein 5 (asb5) and cloned the rabbit full-length cDNA. Asb5 was demonstrated to be a single-copy gene. We localized the asb5 protein in vivo in endothelial and smooth muscle cells of collateral arteries as well as in satellite cells. Asb5 was significantly upregulated in growing collateral arteries on mRNA and protein level. The infusion of doxorubicin in rabbit led to a significant decrease of the asb5 mRNA. In summary, our data show that asb5 is a novel protein implicated in the initiation of arteriogenesis.

Identification of differentially expressed genes like cofilin2 in growing collateral arteries

Arteriogenesis, the growth of pre-existing collateral arteries, can be induced in rabbits by occlusion of the femoral artery. In order to analyze the differential gene expression in arteriogenesis, cDNA of collateral arteries 24h after femoral occlusion or sham operation was subjected to suppression subtractive hybridization (SSH). We demonstrated an upregulation of the U6 snRNA binding protein Lsm5, cytochrome b, an expressed sequence tag, and the actin-depolymerizing factor cofilin2 mRNA in collateral arteries 24h after femoral ligation. For cofilin2, we also detected an increase in the protein level and a localization predominantly in smooth muscle cells of collaterals. Simultaneously with the upregulation of cofilin2 we found a downregulation of the alpha-smooth muscle actin mRNA in growing collateral arteries. In summary, our data showed an augmented expression level of genes contributing to different fundamental processes of arteriogenesis.