Accelerated reendothelialization with suppressed thrombogenic property and neointimal hyperplasia of rabbit jugular vein grafts by adenovirus-mediated gene transfer of C-type natriuretic peptide.

BACKGROUND: Vein graft disease limits the late results of coronary revascularization. C-type natriuretic peptide (CNP) inhibits the growth of vascular smooth muscle cells. Given the effects of CNP on cGMP cascade, we hypothesized that transfected CNP genes modulate endothelial repair and thrombogenicity in the vein graft. METHODS AND RESULTS: Autologous rabbit jugular vein grafts were incubated ex vivo in a solution of adenovirus vectors containing CNP gene (Ad.CNP) or Escherichia coli lac Z gene (Ad.LacZ) and then interposed in the carotid artery. Reendothelialization, mural thrombi formation, and intima/media ratio were evaluated on the 14th and 28th postoperative days. More reendothelialization was seen in Ad.CNP-infected grafts than in Ad.LacZ-infected grafts both at 14 days (0.81+/-0.05 versus 0.30+/-0.14, P<0.01) and at 28 days (0.96+/-0.01 versus 0.45+/-0.08, P<0.001). The mural thrombus area was smaller in Ad.CNP-infected grafts than in Ad.LacZ-infected grafts. Neointimal thickening was significantly suppressed in the Ad.CNP group. The in vitro wound assay with human coronary artery endothelial cells revealed significant potentiation of the wound repair process by CNP and atrial natriuretic peptide administration. CONCLUSIONS: Infected Ad.CNP accelerated reendothelialization and suppressed thrombosis and neointimal hyperplasia. The method may potentially prevent vein graft disease in patients undergoing coronary artery revascularization.

Oxidized LDL regulates vascular endothelial growth factor expression in human macrophages and endothelial cells through activation of peroxisome proliferator-activated receptor-gamma.

Vascular endothelial growth factor (VEGF) has been recognized as an angiogenic factor that induces endothelial proliferation and vascular permeability. Recent studies have also suggested that VEGF can promote macrophage migration, which is critical for atherosclerosis. We have reported that VEGF is remarkably expressed in activated macrophages, endothelial cells, and smooth muscle cells within human coronary atherosclerotic lesions, and we have proposed the significance of VEGF in the progression of atherosclerosis. To clarify the mechanism of VEGF expression in atherosclerotic lesions, we examined the regulation of VEGF expression by oxidized low density lipoprotein (Ox-LDL), which is abundant in atherosclerotic arterial walls. A recent report has revealed that peroxisome proliferator-activated receptor-gamma (PPARgamma) is expressed not only in adipocytes but also in monocytes/macrophages and has suggested that PPARgamma may have a role in the differentiation of monocytes/macrophages. Furthermore, 9- and 13-hydroxy-(S)-10,12-octadecadienoic acid (9- and 13-HODE, respectively), the components of Ox-LDL, may be PPARgamma ligands. Therefore, we investigated the involvement of PPARgamma in the regulation of VEGF by Ox-LDL. PPARgamma expression was detected in human monocyte/macrophage cell lines, human acute monocytic leukemia (THP-1) cells, and human coronary artery endothelial cells (HCAECs). Ox-LDL (10 to 50 microg/mL) upregulated VEGF secretion from THP-1 dose-dependently. VEGF mRNA expression in HCAECs was also upregulated by Ox-LDL. The mRNA expression of VEGF in THP-1 cells and HCAECs was also augmented by PPARgamma activators, troglitazone (TRO), and 15-deoxy-(12,14)-prostaglandin J(2) (PGJ2). In contrast, VEGF expression in another monocyte/macrophage cell line, human histiocytic lymphoma cells (U937), which lacks PPARgamma expression, was not augmented by TRO or PGJ2. We established the U937 cell line, which permanently expresses PPARgamma (U937T). TRO and Ox-LDL augmented VEGF expression in U937T. In addition, VEGF production by THP-1 cells was significantly increased by exposure to 9-HODE and 13-HODE. In conclusion, Ox-LDL upregulates VEGF expression in macrophages and endothelial cells, at least in part, through the activation of PPARgamma.

cGMP-dependent protein kinase phosphorylates and inactivates RhoA.

Small GTPase Rho and cGMP/cGMP-dependent protein kinase (cGK) pathways exert opposing effects in specific systems such as vascular contraction and growth. However, the direct interaction between these pathways has remained elusive. We demonstrate that cGK phosphorylates RhoA in vitro at Ser188, the same residue phosphorylated by cAMP-dependent protein kinase. In HeLa cells transfected with constitutively active cGK (C-cGK), stress fiber formation induced by lysophosphatidic acid or V14RhoA was blocked. By contrast, C-cGK failed to inhibit stress fiber formation in cells transfected with mutant RhoA with substitution of Ser188 to Ala. C-cGK did not affect actin reorganization induced by Rac1 or Rho-associated kinase, one of the effectors for RhoA. Furthermore, C-cGK expression inhibited the membrane translocation of RhoA. Collectively, our findings suggest that cGK phosphorylates RhoA at Ser188 and inactivates RhoA signaling. The physiological relevance of the direct interaction between RhoA and cGK awaits further investigation.

Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors.

Interaction between endothelial cells and mural cells (pericytes and vascular smooth muscle) is essential for vascular development and maintenance. Endothelial cells arise from Flk1-expressing (Flk1+) mesoderm cells, whereas mural cells are believed to derive from mesoderm, neural crest or epicardial cells and migrate to form the vessel wall. Difficulty in preparing pure populations of these lineages has hampered dissection of the mechanisms underlying vascular formation. Here we show that Flk1+ cells derived from embryonic stem cells can differentiate into both endothelial and mural cells and can reproduce the vascular organization process. Vascular endothelial growth factor promotes endothelial cell differentiation, whereas mural cells are induced by platelet-derived growth factor-BB. Vascular cells derived from Flk1+ cells can organize into vessel-like structures consisting of endothelial tubes supported by mural cells in three-dimensional culture. Injection of Flk1+ cells into chick embryos showed that they can incorporate as endothelial and mural cells and contribute to the developing vasculature in vivo. Our findings indicate that Flk1+ cells can act as 'vascular progenitor cells' to form mature vessels and thus offer potential for tissue engineering of the vascular system.

Wavelength-modulated diffraction: a new method for phase determination.

A new diffraction method has been developed in which the intensity of Bragg reflections is measured while changing continually the wavelength of the radiation over a range in the vicinity of the absorption edge of an atom contained in the crystal. It is shown that the intensity gradient with respect to the wavelength of the hkl reflection is in a simple relation to the real and imaginary parts of the structure factor of that reflection and, if the positions of the anomalously scattering atoms are known or properly assumed, the phase of F(hkl) can be derived by solving simultaneous linear equations. The procedure is particularly simple when the crystal is centrosymmetric. The method, called the wavelength-modulated diffraction (WMD) method, is free from the problem of intensity scaling encountered in other methods of phase determination. Synchrotron radiation is most suited to WMD measurements. Suggestions on how to measure the intensity gradient are given and the possible errors involved are discussed.

A compact superconducting ring as a radiation source for X-ray crystallography.

A compact superconducting storage ring installed at Ritsumeikan University is operated at an electron-beam energy of 0.575 GeV and an initial beam current of 300 mA. The radius of the circular electron orbit is as small as 0.5 m, suggesting that the radiation emitted contains short-wavelength components. With an imaging plate as a detector, X-ray precession diffraction patterns were recorded for organic single crystals within a reasonable period of time using radiation of wavelength 0.155 nm (8 keV) to 0.248 nm (5 keV). The use of the radiation in the structural study of organic crystals containing 3d metal atoms using the phenomena of anomalous scattering is described. If appropriately planned, X-ray diffraction and/or scattering experiments can be made at the compact ring without recourse to a large-scale ring.

Relation of certain infrared bands to conformational changes of cellulose and cellulose oligosaccharides.

The i.r. spectra of D-glucose and cellulose oligosaccharides up to cellopentaose have been compared with those of cellulose at various temperatures between that of liguid nitrogen and approximately 250 degrees. Significant changes in frequency and intensity of the bands at approximately 3400 cm -1 were observed. The a 1372 cm -1/a2900 cm -1 ratio for each carbohydrate studied decreased gradually as the temperature was increased above ambient. The change of the band intensities at 1429 and 893 cm -1 with temperature was also investigated. The observed spectral changes are assumed to be associated with changes of hydrogen bonding.