Frederic Pipp

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.

The Range of Adaptation by Collateral Vessels After Femoral Artery Occlusion

Natural adaptation to femoral artery occlusion in animals by collateral artery growth restores only ≈35% of adenosine-recruitable maximal conductance (Cmax) probably because initially elevated fluid shear stress (FSS) quickly normalizes. We tested the hypothesis whether this deficit can be mended by artificially increasing FSS or whether anatomical restraints prevent complete restitution. We chronically increased FSS by draining the collateral flow directly into the venous system by a side-to-side anastomosis between the distal stump of the occluded femoral artery and the accompanying vein. After reclosure of the shunt collateral flow was measured at maximal vasodilatation. Cmax reached 100% already at day 7 and had, after 4 weeks, surpassed (2-fold) the Cmax of the normal vasculature before occlusion. Expression profiling showed upregulation of members of the Rho-pathway (RhoA, cofilin, focal adhesion kinase, vimentin) and the Rho-antagonist Fasudil markedly inhibited arteriogenesis. The activities of Ras and ERK-1,-2 were markedly increased in collateral vessels of the shunt experiment, and infusions of L-NAME and L-NNA strongly inhibited MAPK activity as well as shunt-induced arteriogenesis. Infusions of the peroxinitrite donor Sin-1 inhibited arteriogenesis. The radical scavengers urate, ebselen, SOD, and catalase had no effect. We conclude that increased FSS can overcome the anatomical restrictions of collateral arteries and is potentially able to completely restore maximal collateral conductance. Increased FSS activates the Ras-ERK-, the Rho-, and the NO- (but not the Akt-) pathway enabling collateral artery growth.

Elevated Fluid Shear Stress Enhances Postocclusive Collateral Artery Growth and Gene Expression in the Pig Hind Limb

Objective—The role of fluid shear stress (FSS) in collateral vessel growth remains disputed and prospective in vivo experiments to test its morphogenic power are rare. Therefore, we studied the influence of FSS on arteriogenesis in a new model with extremely high levels of collateral flow and FSS in pig and rabbit hind limbs. Methods and Results—A side-to-side anastomosis was created between the distal stump of one of the bilaterally occluded femoral arteries with the accompanying vein. This clamps the collateral reentry pressure at venous levels and increases collateral flow, which is directed to a large part into the venous system. This decreases circumferential wall stress and markedly increases FSS. One week after anastomosis, angiographic number and size of collaterals were significantly increased. Maximal collateral flow exceeded by 2.3-fold that obtained in the ligature-only hind limb. Capillary density increased in lower leg muscles. Immunohistochemistry revealed augmented proliferative activity of endothelial and smooth muscle cells. Intercellular adhesion molecule-1 and vascular cell adhesion molecule (VCAM)-1 were upregulated, and monocyte invasion was markedly increased. In 2-dimensional gels, actin-regulating cofilin1 and cofilin2, destrin, and transgelin2 showed the highest degree of differential regulation. Conclusions—High levels of FSS cause a strong arteriogenic response, reinstate cellular proliferation, stimulate cytoskeletal rearrangement, and normalize maximal conductance. FSS is the initiating molding force in arteriogenesis.

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

OBJECTIVE Collateral 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. METHODS 24 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. RESULTS We 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. CONCLUSIONS Our 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.

Porcine aortic endothelial cells show little effects on smooth muscle cells but are potent stimulators of cardiomyocyte growth

Smooth muscle cells (SMC) and endothelial cells (EC) play a pivotal role in arteriogenesis and atherosclerosis. We evaluated the role of EC on the growth of SMC and neonatal cardiomyocytes (NEO) by using serum-free EC-supernatant (AoCM). Five percent fetal calf serum was used in order to mimic growth effects of blood. EC and SMC purities were 99% as determined by absence or presence of markers such as CD31, desmin, α-smooth muscle actin and tropomyosin using immunostaining and FACS analysis. AoCM markedly influenced the morphology of NEO as determined by α-actinin staining but showed only little effect on the phenotype of SMC. Protein synthesis after 2 days increased 2.5-fold in SMC and 3.7-fold in NEO as determined by tritium incorporation. The values for serum (2.8 and 2.3-fold, respectively) were comparable. The induction of DNA-synthesis by serum in NEO was twice that of AoCM (3.9-fold). However, proliferative effects of serum and AoCM on SMC differed markedly: Serum induced a 66-fold increase in DNA-synthesis resulting in a 54% higher cell number. DNA-synthesis after AoCM treatment lead to a nonsignificant small increase and no proliferation was detected. Platelet derived growth factor (PDGF-AB), present in blood, induced a 47-fold increase in DNA-synthesis and a 38% increase in cell number. Our data suggest that EC in the absence of physical forces exert strong morphogenic effects on cardiomyocytes but they lack specific effects on smooth muscle cells. In vessels EC might function as a border to isolate SMC from key regulators in blood such as PDGFs.

Dissection of Monocyte and Endothelial Activities by Using VEGF-Receptor Specific Ligands

Vascular endothelial growth factor (VEGF) is the major inducer of angiogenesis and vasculogenesis (Risau, 1997). It was isolated based on its ability to induce proliferation of endothelial cells but not fibroblasts (Leung et al., 1989). Based on this competency VEGF emerged as a highly good candidate as an angiogenesis-specific factor. Because VEGF is produced in response to hypoxia it describes a physiological mean to ablate the need of nutrients and oxygen by the induction of new blood vessels. In vitro, it induces several activities in endothelial cells, which are believed to be associated with angiogenesis, such as proliferation, survival and migration. But it also displays activities in endothelial cells, which were different from what was expected from an endothelial cell specific mitogen. VEGF can also induce vascular hyperpermeability, leading to its original description as vascular permeability factor (VPF) and turned out to be an inducer of tissue factor, the initiator of blood coagulation (Nemerson, 1988). In addition it is able to increase both the plasminogen activator and its inhibitor (Pepper et al., 1991). VEGF was found to cause release of von Willebrand factor from the Weibel-Palade bodies in endothelial cells and to increase the surface expression of P-selectin, two processes which comprise possible links to blood coagulation and inflammation, respectively. In consequence, the question arose whether VEGF would be a jack of all trades, comparable to another unspecific growth factor, fibroblast growth factor (FGF) (Clauss and Schaper, 2000). This point of view was enforced by the early finding that VEGF not only acts on endothelial cells but also on other cells. In this context monocytes were identified shortly after the discovery of VEGF as vascular endothelial growth factor (Clauss et al., 1990). In monocytes, VEGF induces chemotaxis, transmigration through endothelial monolayers, tissue factor and the inducible NO-Synthetase (Clauss, 1998). Furthermore, it was found to inhibit the differentiation to dendritic cells and to enforce the transition to endothelial cells (Gabrilovich et al., 1998). These diverse activities are not necessarily associated with angiogenesis. It should therefore be important to understand the mechanism of VEGF-elicited activities and, if possible to be able to distinguish VEGF-mediated activities on endothelial cells from those onto monocytes.

Rapid Identification of Differentially Expressed Genes by Combination of SSH and MOS

uppression subtractive hybridization (SSH) is atechnique especially designed for the detection ofrare transcripts, which vary in their expression patternbetween two experimental setups (Diatchenko et al,1996). SSH, which allows a synchronous normaliza-tion and subtraction of two cDNA pools in one step,shows several advantages compared with other tech-niques. In contrast to DNA-microarrays, SSH does notrely on the availability of specific sequences. Likedifferential display, SSH also allows the identificationof unknown genes, but the identified cDNA fragmentsare longer and often located in the coding region,thereby facilitating the assignment to a certain gene.One drawback of SSH, however, is the occurrence ofbackground molecules in the subtracted library. Theycan arise via unspecific annealing during the ligation ofthe adaptor-molecules or represent redundant cDNAfragments that were not subtracted during the twohybridizations. These cDNAs give positive results inthe first screening process, but their differential ex-pression cannot be confirmed by independent tech-niques like Northern blot hybridizations. Mirror orien-tation selection (MOS) (Rebrikov et al, 2000) makesuse of the fact that background molecules are notamplified by PCR and therefore have only one relativeorientation to the adaptor molecule. Target genes,however, have many predecessor molecules as aresult of the amplification, and are represented byboth orientations relative to the adaptor. The technicalprocedure involves the detachment of one adaptor,heat-denaturation, and re-annealing of the sample, aswell as an exponential amplification of the targetmolecules by PCR. We describe a shortening andsimplification of the screening process for differentiallyexpressed genes in subtracted libraries making use ofslot-blot analysis and the elimination of backgroundmolecules by MOS.Differential gene expression was studied via SSH ina rabbit model of arteriogenesis (growth of pre-existing collateral arteries) (Ito et al, 1997). Collateralarteries were isolated from the surrounding muscletissue 24 hours after induction of arteriogenesis byfemoral artery occlusion as well as of sham-operatedanimals. Total RNA was isolated as described previ-ously (Chomczynski and Sacchi, 1987) and 10 gwere treated with DNase. The mRNA was extracted(Oligotex Mini Kit, Qiagen, Hilden, Germany), amplifiedusing the SMART-technique (SMART PCR cDNA Syn-thesis Kit, BD-Clontech, Palo Alto, California), andsubjected to SSH (PCR Select cDNA Subtraction Kit,BD-Clontech). In the forward hybridization, the cDNAderived from collateral arteries 24 hours after femoralocclusion served as tester and the cDNA of collaterals24 hours after sham operation as driver (and viceversa for the reverse subtraction). The subtractionswere performed according to the manufacturers pro-tocol. PCR products were cloned in pGEM-Teasy(Promega, Madison, Wisconsin). To screen for differ-entially expressed genes we performed slot-blot anal-ysis, hybridizing clones from the forward subtractedlibrary with forward and reverse subtracted cDNApools.Bacteria transformed with cDNA fragments of theforward subtracted library were grown in 100 lLB ampicillin at 37° C overnight and 1 volume ofphenol/chloroform was added. After mixing and cen-trifugation (14,000 g, 2 minutes) the aqueous phasewas transferred to a fresh tube. Saline sodium citrate, 20 (SSC; 3 M NaCl; 0.3 M sodium citrate), wasadded to a concentration of 6 SSC in a total volumeof 400 l. The samples were boiled for 10 minutes andcooled on ice. A total of 175 l of the solutions,corresponding to approximately 5 ng plasmid-DNA,were applied to nylon membranes (Duralon UV mem-brane, Stratagene, La Jolla, California) using a slot-blot apparatus. Two identical filters were created,which were incubated for 15 minutes in denaturingsolution (1.5 M NaCl; 0.5 M NaOH), for 15 minutes inrenaturing solution (1.5 M NaCl; 0.5 M Tris/HCl pH8.0), and UV-crosslinked (UV-Stratalinker, Strat-