Micheline Duriez

Lactadherin promotes VEGF-dependent neovascularization

Vascular endothelial growth factor (VEGF)-induced blood vessel growth is involved in both physiological and pathological angiogenesis and requires integrin-mediated signaling. We now show that an integrin-binding protein initially described in milk-fat globule, MFG-E8 (also known as lactadherin), is expressed in and around blood vessels and has a crucial role in VEGF-dependent neovascularization in the adult mouse. Using neutralizing antibodies and lactadherin-deficient animals, we show that lactadherin interacts with αvβ3 and αvβ5 integrins and alters both VEGF-dependent Akt phosphorylation and neovascularization. In the absence of VEGF, lactadherin administration induced αvβ3- and αvβ5-dependent Akt phosphorylation in endothelial cells in vitro and strongly improved postischemic neovascularization in vivo. These results show a crucial role for lactadherin in VEGF-dependent neovascularization and identify lactadherin as an important target for the modulation of neovascularization.

Impairment in Ischemia-Induced Neovascularization in Diabetes: Bone Marrow Mononuclear Cell Dysfunction and Therapeutic Potential of Placenta Growth Factor Treatment

Mechanisms that hinder ischemia-induced neovascularization in diabetes remain poorly understood. We hypothesized that endogenous bone marrow mononuclear cell (BM-MNC) dysfunction may contribute to the abrogated postischemic revascularization reaction associated with diabetes. We first analyzed the effect of diabetes (streptozotocin, 40 mg/kg) on BM-MNC pro-angiogenic potential in a model of surgically induced hindlimb ischemia. In nondiabetic animals, transplantation of BM-MNCs isolated from nondiabetic animals raised the ischemic/nonischemic angiographic score, capillary number, and blood flow recovery by 1.8-, 2.7-, and 2.2-fold, respectively, over that of PBS-injected nondiabetic animals (P < 0.05). Administration of diabetic BM-MNCs also improved the neovascularization reaction in ischemic hindlimbs of nondiabetic mice but to a lesser extent from that observed with nondiabetic BM-MNC transplantation. In diabetic mice, injection of nondiabetic BM-MNCs was still more efficient than that of diabetic BM-MNCs. Such BM-MNC dysfunction was associated with the impairment of diabetic BM-MNC capacity to differentiate into endothelial progenitor cells (EPCs) in vitro and to participate in vascular-like structure formation in a subcutaneous Matrigel plug. Placenta growth factor (PlGF) administration improved by sixfold the number of EPCs differentiated from diabetic BM-MNCs in vitro and enhanced ischemic/nonischemic angiographic score, capillary number and blood flow recovery by 1.9-, 1.5- and 1.6-fold, respectively, over that of untreated diabetic animals (P < 0.01). Endogenous BM-MNC pro-angiogenic potential was affected in diabetes. Therapeutic strategy based on PlGF administration restored such defects and improved postischemic neovascularization in diabetic mice.

Antiangiogenic Effect of Interleukin-10 in Ischemia-Induced Angiogenesis in Mice Hindlimb

Ischemia induces both hypoxia and inflammation that trigger angiogenesis. The inflammatory reaction is modulated by production of anti-inflammatory cytokines. This study examined the potential role of a major anti-inflammatory cytokine, interleukin (IL)–10, on angiogenesis in a model of surgically induced hindlimb ischemia. Ischemia was produced by artery femoral occlusion in both C57BL/6J IL-10+/+ and IL-10–/– mice. After 28 days, angiogenesis was quantified by microangiography, capillary, and arteriole density measurement and laser Doppler perfusion imaging. The protein levels of IL-10 and vascular endothelial growth factor (VEGF) were determined by Western blot analysis in hindlimbs. IL-10 was markedly expressed in the ischemic hindlimb of IL-10+/+ mice. Angiogenesis in the ischemic hindlimb was significantly increased in IL-10–/– compared with IL-10+/+ mice. Indeed, angiographic data showed that vessel density in the ischemic leg was 10.2±0.1% and 5.7±0.4% in IL-10–/– and IL-10+/+ mice, respectively (P <0.01). This corresponded to improved ischemic/nonischemic leg perfusion ratio by 1.4-fold in IL-10–/– mice compared with IL-10+/+ mice (0.87±0.05 versus 0.63±0.01, respectively;P <0.01). Revascularization was associated with a 1.8-fold increase in tissue VEGF protein level in IL-10–/– mice compared with IL-10+/+ mice (P <0.01). In vivo electrotransfer of murine IL-10 cDNA in IL-10–/– mice significantly inhibited both the angiogenic process and the rise in VEGF protein level observed in IL-10–/– mice. No changes in vessel density or VEGF content were observed in the nonischemic hindlimb. These findings underscore the antiangiogenic effect of IL-10 associated with the downregulation of VEGF expression and suggest a role for the inflammatory balance in the modulation of ischemia-induced angiogenesis.

Cardiac functional improvement by a human Bcl-2 transgene in a mouse model of ischemia/reperfusion injury.

Apoptosis has been shown to contribute to myocardial reperfusion injury. It has been suggested that, in reducing the apoptotic component within the ischemic area at risk, Bcl‐2 overexpression could lead to a ventricular function improvement.

Vascular Endothelial Growth Factor-B Promotes In Vivo Angiogenesis

&NA; Vascular endothelial growth factors (VEGFs) and their receptors have emerged as central regulators of the angiogenic process. However, involvement of VEGF‐B, one of these factors, in angiogenesis remains obscure. Mice received subcutaneous injection of Matrigel alone or Matrigel with human recombinant protein rhVEGF‐B167 or with rhVEGF‐A165. After 14 days, cell ingrowth in the Matrigel plug was increased by 2.0‐ and 2.5‐fold in rhVEGF‐B167‐treated and rhVEGF‐A165‐treated mice, respectively (P<0.01), in association with a raise in phospho‐Akt/Akt (1.8‐fold, P<0.01) and endothelial NO synthase (eNOS) (1.80‐ and 1.60‐fold, respectively; P<0.05) protein levels measured by Western blot. VEGF‐B‐induced cell ingrowth was impaired by treatment with NOS inhibitor (NG‐nitro‐L‐arginine methyl ester; L‐NAME, 10 mg/kg per day). Treatment with neutralizing antibody directed against the VEGF‐B receptor VEGF‐R1 (anti‐VEGFR1, 10 &mgr;g) completely abrogated VEGF‐B‐related effects. Proangiogenic effect of VEGF‐B was confirmed in a mouse model of surgically induced hindlimb ischemia. Plasmids containing human form of VEGF‐A (phVEGF‐A165) or VEGF‐B (phVEGF‐B167 or phVEGF‐B186) were administered by in vivo electrotransfer. Angiographic score at day 28 showed significant improvement in ischemic/nonischemic leg ratio by 1.4‐ and 1.5‐fold in mice treated with phVEGF‐B167 and phVEGF‐B186, respectively (P<0.05). Laser Doppler perfusion data also evidenced a 1.5‐fold increase in phVEGF‐B167‐treated and phVEGF‐B186‐treated mice (P<0.05). Such an effect was associated with an upregulation of phospho‐Akt/Akt and eNOS protein levels in the ischemic legs and was hampered by treatment with anti‐VEGFR1. This study demonstrates for the first time that VEGF‐B, in part through its receptor VEGF‐R1, promotes angiogenesis in association with an activation of Akt and eNOS‐related pathways. (Circ Res. 2003;93:114‐123.)

Angiotensin converting enzyme inhibition prevents the increase in aortic collagen in rats.

Four groups of 4-week-old spontaneously hypertensive rats were treated during 4 months with the angiotensin converting enzyme inhibitor quinapril at 1 mg/kg per day (Q1) or 10 mg/kg per day (Q10), hydralazine at 15 mg/kg per day (H), or placebo (P). In the first set of experiments, blood pressure was measured in conscious rats, and plasma and aortic angiotensin converting enzyme activities were evaluated. In the second set of experiments, histomorphometric parameters of the thoracic aorta were evaluated. Mean blood pressure was lower in the Q10 and H groups (136 +/- 16 and 149 +/- 11 mm Hg) compared with the P group (190 +/- 23 mm Hg) (P < .01). The Q1 group showed mean blood pressure values (171 +/- 15 mm Hg) lower than the P group (P < .05) but significantly higher than the Q10 and H groups (P < .01 and P < .05, respectively). Aortic medial cross-sectional area was significantly lower in the H and Q10 groups (455 +/- 61 and 487 +/- 57 x 10(3) microns 2) than in the P group (636 +/- 72 x 10(3) microns 2) (P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)

CD40 ligand+ microparticles from human atherosclerotic plaques stimulate endothelial proliferation and angiogenesis a potential mechanism for intraplaque neovascularization

OBJECTIVES Our goal was to demonstrate that microparticles (MPs) are the endogenous signal leading to neovessel formation through CD40 ligation in human atherosclerotic plaques. BACKGROUND Vulnerable atherosclerotic plaques prone to rupture are characterized by an increased number of vasa vasorum and frequent intraplaque hemorrhage. Although inflammatory cytokines, growth factors, or CD40/CD40 ligand (CD40L) are possible candidates, the mechanism of atherosclerotic plaque neovascularization remains unknown. Atherosclerotic plaques contain large amounts of membrane-shed submicron MPs released after cell activation or apoptosis. METHODS Microparticles were isolated from endarterectomy specimens surgically obtained from 26 patients and characterized by phosphatidylserine exposure and specific markers of cellular origin. RESULTS Plaque MPs increased both endothelial proliferation assessed by (3)H-thymidine incorporation and cell number and stimulated in vivo angiogenesis in Matrigel (BD Biosciences, San Diego, California) assays performed in wild-type and BalbC/Nude mice, whereas circulating MPs had no effect. Microparticles from symptomatic patients expressed more CD40L and were more potent in inducing endothelial proliferation, when compared with asymptomatic plaque MPs. Most of CD40L+ MPs (93%) isolated from human plaques were of macrophage origin. Microparticle-induced endothelial proliferation was impaired by CD40L or CD40-neutralizing antibodies and abolished after endothelial CD40-ribonucleic acid silencing. In addition, the proangiogenic effect of plaque MPs was abolished in Matrigel assays performed in the presence of CD40L-neutralizing antibodies or in CD40-deficient mice. CONCLUSIONS These results demonstrate that MPs isolated from human atherosclerotic lesions express CD40L, stimulate endothelial cell proliferation after CD40 ligation, and promote in vivo angiogenesis. Therefore, MPs could represent a major determinant of intraplaque neovascularization and plaque vulnerability.

Transplantation of Bone Marrow–Derived Mononuclear Cells in Ischemic Apolipoprotein E–Knockout Mice Accelerates Atherosclerosis Without Altering Plaque Composition

Background—Bone marrow–derived mononuclear cells (BM-MNCs) enhance postischemic neovascularization, and their therapeutic use is currently under clinical investigation. We evaluated the safety of BM-MNC–based therapy in the setting of atherosclerosis. Methods and Results—Apolipoprotein E (apoE)–knockout (KO) mice were divided into 4 groups: 20 nonischemic mice receiving intravenous injection of either saline (n=10) or 106 BM-MNCs from wild-type animals (n=10) and 20 mice with arterial femoral ligature receiving intravenous injection of either saline (n=10) or 106 BM-MNCs from wild-type animals (n=10) at the time of ischemia induction. Animals were monitored for 4 additional weeks. Atherosclerosis was evaluated in the aortic sinus. BM-MNC transplantation improved tissue neovascularization in ischemic hind limbs, as revealed by the 210% increase in angiography score (P <0.0001), the 33% increase in capillary density (P =0.01), and the 65% increase in tissue Doppler perfusion score (P =0.0002). Hindlimb ischemia without BM-MNC transplantation or BM-MNC transplantation without ischemia did not affect atherosclerotic plaque size. However, transplantation of 106 BM-MNCs into apoE-KO mice with hindlimb ischemia induced a significant 48% to 72% increase in lesion size compared with the other 3 groups (P =0.0025), despite similar total cholesterol levels. Transplantation of 105 BM-MNCs produced similar results, whereas transplantation of 106 apoE-KO–derived BM-MNCs had neither proangiogenic nor proatherogenic effects. There was no difference in plaque composition between groups. Conclusions—BM-MNC therapy is unlikely to affect atherosclerotic plaque stability in the short term. However, it may promote further atherosclerotic plaque progression in an ischemic setting.

Blockade of advanced glycation end-product formation restores ischemia-induced angiogenesis in diabetic mice

We hypothesized that formation of advanced glycation end products (AGEs) associated with diabetes reduces matrix degradation by metalloproteinases (MMPs) and contributes to the impairment of ischemia-induced angiogenesis. Mice were treated or not with streptozotocin (40 mg/kg) and streptozotocin plus aminoguanidine (AGEs formation blocker, 50 mg/kg). After 8 weeks of treatment, hindlimb ischemia was induced by right femoral artery ligature. Plasma AGE levels were strongly elevated in diabetic mice when compared with control mice (579 ± 21 versus 47 ± 4 pmol/ml, respectively; P < 0.01). Treatment with aminoguanidine reduced AGE plasma levels when compared with untreated diabetic mice (P < 0.001). After 28 days of ischemia, ischemic/nonischemic leg angiographic score, capillary density, and laser Doppler skin-perfusion ratios were 1.4-, 1.5-, and 1.4-fold decreased in diabetic mice in reference to controls (P < 0.01). Treatment with aminoguanidine completely normalized ischemia-induced angiogenesis in diabetic mice. We next analyzed the role of proteolysis in AGE formation-induced hampered neovascularization process. After 3 days of ischemia, MMP-2 activity and MMP-3 and MMP-13 protein levels were increased in untreated and aminoguanidine-treated diabetic mice when compared with controls (P < 0.05). Despite this activation of the MMP pathway, collagenolysis was decreased in untreated diabetic mice. Conversely, treatment of diabetic mice with aminoguanidine restored collagenolysis toward levels found in control mice. In conclusion, blockade of AGE formation by aminoguanidine normalizes impaired ischemia-induced angiogenesis in diabetic mice. This effect is probably mediated by restoration of matrix degradation processes that are disturbed as a result of AGE accumulation.

Antiangiogenic Effect of Angiotensin II Type 2 Receptor in Ischemia-Induced Angiogenesis in Mice Hindlimb

This study examined the potential role of angiotensin type 2 (AT2) receptor on angiogenesis in a model of surgically induced hindlimb ischemia. Ischemia was produced by femoral artery ligature in both wild-type and AT2 gene–deleted mice (Agtr2−/Y). After 28 days, angiogenesis was quantitated by microangiography, capillary density measurement, and laser Doppler perfusion imaging. Protein levels of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), Bax, and Bcl-2 were determined by Western blot analysis in hindlimbs. The AT2 mRNA level (assessed by semiquantitative RT-PCR) was increased in the ischemic hindlimb of wild-type mice. Angiographic vessel density and laser Doppler perfusion data showed significant improvement in ischemic/nonischemic leg ratio, 1.9- and 1.7-fold, respectively, in Agtr2−/Y mice compared with controls. In ischemic leg of Agtr2−/Y mice, revascularization was associated with an increase in the antiapoptotic protein content, Bcl-2 (211% of basal), and a decrease (60% of basal) in the number of cell death, determined by TUNEL method. Angiotensin II treatment (0.3 mg/kg per day) raised angiogenic score, blood perfusion, and both VEGF and eNOS protein content in ischemic leg of wild-type control but did not modulate the enhanced angiogenic response observed in untreated Agtr2−/Y mice. Finally, immunohistochemistry analysis revealed that VEGF was mainly localized to myocyte, whereas eNOS-positive staining was mainly observed in the capillary of ischemic leg of both wild-type and AT2-deficient mice. This study demonstrates for the first time that the AT2 receptor subtype may negatively modulate ischemia-induced angiogenesis through an activation of the apoptotic process.