The Mechanism Of Hyperhomocysteinimia Impaired Angiogenesis

Abundant epidemiological and clinical studies have revealed the close relationship between hyperhomocysteinemia (HHcy) and the increased frequency of CVD. Meta-analysis has also shown that an increase of5μmol/L in plasma homocysteine (Hcy) levels enhances the risk of CVD by1.6-to1.8-fold.During the last decade, we and others have demonstrated that HHcy can inhibit endothelial cell growth and postinjury reendothelialization, accelerate neointimal formation, also can impair endothelial relaxation, stimulate vascular smooth muscle cell proliferation, and inhibit high-density lipoprotein biosynthesis. However, the fundamental basis of HHcy-impaired angiogenesis remains unknown.Part I HHcy impair mouse angiogenesisObject:To verify whether HHcy impair mouse angiogenesis by using these classic angiogenesis models.Method:1. Cystathionine-synthase (CBS) is a key enzyme in the transsulfuration pathway that catabolizes Hcy. Kruger’s laboratory (Fox Chase Cancer Center, Philadelphia, PA) created a transgenic mouse (Tg-hCBS) in which the human CBS cDNA is under the control of a zinc-inducible metallothionein promoter. By crossing these animals to Cbs mice and supplying zinc in the drinking water during pregnancy and lactation, Tg-hCBS was able to rescue the neonatal lethal phenotype and generated Tg-hCBS+Cbs-/-animals. At weaning, zinc was removed and the expression of Tg-hCBS was stopped, which caused the animals to develop severe HHcy (plasma Hcy>115μmol/L).2. Verify the influence of Hcy on angiogenesis.2.1Mouse eyeballs were dissected from postnatal day1,3,7,14mice and8-12weeks old Tg-hCBS+Cbs+/+, Tg-hCBS+Cbs+/-, and Tg-hCBS+Cbs-/-mice. Mouse retinals were cut into four radial incisions and whole-mount was staining of Isolectin B4(endothelial cell). Images were acquired by2-laser Nikon confocal microscopy.3-dimensiona image of the whole retinal vascular plexus and each layer of plexus were taken.2.2Aortic ring assay. Sacrifice the8-12month old mice and excise thoracic aorta. Transfer the aorta into a dish with sterilized1x PBS buffer. After removing the fibroadipose tissue, section arteries into1-1.5mm long cross sections, rinse the sections five times with endothelial cell growth medium (ECGM), and place them on the Matrigel-coated wells. Cover a48-well plate with Matrigel (100μl/well) and incubate at37℃,5%CO2concentration for30min previously. Covered the artery rings with an additional100μl of Matrigel. Incubate the rings in1.5ml of ECGM medium。Take images of aortic rings everyday. Delineate the outgrowth area and measure the outgrowth micro-vessel with the HCA-Vision software (Bio Image).Result:1. HHcy impairs mouse angiogenesis in the retinal of hCBS+Cbs-/-mice. Retinal vasculature plexus length of both postnatal (day1,3,7,14) and adult (12weeks old) hCBS+Cbs-/-mice are all decreased.2. Hcy suppresses mice aortic ring micro-vessel growth. Micro-vessel growth was impaired in day5during in-vitro culture of HHcy (hCBS+Cbs-/-) mice. Hcy impaired aorta ring micro-vessel growth in a concentration-dependent manner.Conclusion:HHcy impair angiogenesis in hCBS+Cbs-/-mice. Part II HHcy impair angiogenesis via HGF inhibitionObject:The molecular mechanism of HHcy impaird angiogenesis Method:1. Tube formation. The HAEC/HCMEC were seeded on growth factor-reduced Matrigel and visualized8-12h later, in control conditions, or in the presence of100/200/500μm DL-Hcy. Compare the tube length and tube loop number between two groups.2. The Human angiogenesis RT2ProfilerTMPCR Array profiles the expression of84key genes involved in modulating the biological processes of angiogenesis. The array includes growth factors and their receptors, chemokines and cytokines, matrix and adhesion molecules, proteases and their inhibitors, as well as transcription factors, all involved in the development of new blood vessels. Using Real-time PCR, we analyze expression of a focused panel of genes related to angiogenesis with this array.3. Extract RNA and protein from HAEC/mouse aorta/mouse heart tissue, Real-time PCR and western blot to confirm the down-regulate of Hepatocyte Growth Factor (HGF) under Hcy treat or in HHcy mice.4. Set up acute myocardial infarction (MI) model. A permanent knot was made around the left anterior descending coronary artery (LAD)2-3mm from its origin with a6-0silk suture. Sham-operated animals were subjected to the same surgical procedures except that the suture was passed under the LAD but was not tied. Sacrifice the mice two weeks later and collect heart tissue. Extract protein from mice tissues as described before. Compare HGF expression level between two groups.5. Mouse HGF Enzyme Linked Immunosorbent Assay (ELISA) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of mouse HGF in serum. This assay employs an antibody specific for mouse HGF coated on a96-well plate and the intensity of the color is measured at450nm.6. HGF repair Hcy impaird angiogenesis. Add HGF back into tube formation and aorta ring assay, check tube length and micro-vessel growth.Result:1. Hcy impaird HAEC/HCMEC in vitro tube formation process in a concentration-dependent manner.2. Angiogenesis RT2ProfilerTMPCR Array revealed up-and down-regulated genes in HAEC. Total RNA from HAEC control group and Hcy group were characterized in technical triplicates. Genes encoding the Collagen, type IV, alpha3(COL4A3), Epiregulin (EREG), Hepatocyte growth factor (HGF), Interferon (IFNB1&IFNG), and Leukocyte cell derived chemotaxin1(LECT1) were down-regulated, by at least four-fold (outside the blue field) in Hcy group relative to control group.3. HAEC/Aorta/Heart of hCBS+Cbs+/-and hCBS+Cbs-/-mice were collected at age of8-12weeks. HGF and β-actin mRNA were detected by RT-PCR, HGF and β-actin proteins were detected by western blot. Quantitative results are shown that Hcy impair HAEC/Aorta/Heart HGF expression.4. We established acute myocardial infarction (MI) in hCBS+Cbs+/-and hCBS+Cbs-/-mice by left coronary artery (LAD) ligation. Hearts were collected at2weeks after surgery. HGF and β-actin protein expression were detected by western blot. Quantitative results are also shown that HHcy impair heart HGF expression under MI.5. HGF level in mouse serum was detected by Enzyme-Linked Immunosorbent Assay (ELISA). Standards and samples are pipetted into the96-wells. The intensity of the color is measured at450nm. HGF decreased in hCBS+Cbs-/-mice serum and also decreased in hCBS+Cbs-/-+MI mice serum.6. HGF repair Hcy inhibited tube formation and aorta ring assay. Tube length and micro-vessel are all increased after HGF treat.Conclustion:HHcy impair angiogenesis via HGF inhibition Part Ⅲ Endothelial progenitor cell join in HHcy impaired angiogenesisObject:Role of endothelial progenitor cell in HHcy impaired angiogenesis. Intravenous transfusions of Sca-1+cells play a role in HHcy MI mice angiogenesis. Method:1. Angiogenesis of HHcy mice under myocardial infarction.1.1Cardiac function was measured with echocardiography (VisualSonics Vevo770). Hearts were viewed in the short-axis between the two papillary muscles and analyzed in M-mode. Parameters (Vevo software) including end-diastolic diameter (EDD), end-systolic diameter (ESD), posterior wall thickness (PWT), and sepal wall thickness (SWT) were measured to determine changes in cardiac morphology and function (ejection fraction (EF) and fractional shortening (FS));1.2Hearts were moved at2weeks/6weeks after myocardial infarction and kept at-80℃until needed. Frozen heart tissues were cut into5μm thick slices. Adjacent sections (taken at the midpoint between LAD ligation site and apex) were stained with rabbit polyclonal antibodies against CD31. Capillary density was expressed as CD31+endothelial cells per high-power field (200x).2. Flow cytometry analysis. A volume of200μl peripheral blood/bone marrow were incubated for30minutes in the dark with monoclonal antibodies against mouse vascular endothelial growth factor receptor2(VEGFR2) followed by PE-conjugated secondary antibody, with the APC-labeled monoclonal antibodies against mouse Stem cell antigen-1(Sca-1). Isotypeidentical antibodies served as controls. After incubation, cells were washed with PBS, and fixed in2%paraformaldehyde before analysis. Each analysis included100000events.3. MACS Separation-purify Sca-1+cells. Purity of Sca-1+cell is based on the use of MACS MicroBeads, MACS Columns and MACS Separators. As MACS MicroBeads are extremely small, superparamagnetic particles, a high-gradient magnetic field is required to retain the labeled cells. Cells are initially immunolabeled with Anti-Sca-1-APC, after which magnetic labeling of Sca-1+cells can be achieved using Anti-APC MicroBeads. The cell suspension is then applied to a MACS(?)olumn placed in the magnetic field of a MACS Separator. The magnetically labeled Sca-1+cells are retained within the column, while the unlabeled Sca-1-cells are eluted in the flow through. After removal of the column from the magnetic field the retained Sca-1+cells are eluted. Result:1. HHcy impairs mouse cardiac function. Ejection fraction (EF) and fractional shortening (FS) were lower in HHcy mice group than control group, as well as heart capillary density. HHcy mice hearts have depressed function and less capillary density after myocardial infarction stress. Survival rate is lower in HHcy mice.2. Peripheral blood derived EPC percentage decreased in HHcy mice group and bone marrow derived EPC percentage is higher in HHcy mice group, but cell death rate is also higher in HHcy mice.3. Intravenous transfusion of Sca-1+cells treatment induce PB derived EPC percentage increase in both control mice group and HHcy mice group. Survival rate and heart function also increase after cell treatment.Conclusion:EPC joined angiogenesis after myocardial infarction which is so important to cardiac function. Cell treatment restores ischemia-induced angiogenesis in HHcy mice.