Chaoyong Zhu

Expression of Neutrophil Gelatinase–Associated Lipocalin in Atherosclerosis and Myocardial Infarction

Objective—Neutrophil gelatinase-associated lipocalin (NGAL) modulates the activity of matrix metalloproteinase (MMP) 9, an important mediator of vascular remodeling and plaque instability in atherosclerosis. This study aimed to analyze the expression of NGAL in atherosclerotic plaques and myocardial infarction (MI). Methods and Results—Atherosclerotic apolipoprotein E (apoE)−/− × low-density lipoprotein receptor (LDLR)−/− and C57BL/6J control mice were exposed to brief hypoxic stress (10 minutes of 10% oxygen). Expression of the mouse NGAL homolog (24p3) and MMP-9 was analyzed 48 hours later by quantitative RT-PCR, immunohistochemistry, and zymography. Hypoxic stress increased NGAL/24p3 mRNA in the cardiac vasculature. NGAL/24p3 was also increased in atherosclerotic plaques of apolipoprotein E−/− × LDLR−/− mice compared with C57BL/6J mice. Mice developing MI exhibited the highest plaque mRNA expression of NGAL/24p3 and MMP-9. Zymography revealed strong proteolytic activity in areas rich in 24p3 and MMP-9 protein. Immunohistochemistry performed on human carotid endarterectomy specimens and control tissue from the internal mammary artery showed colocalization of MMP-9 and NGAL with macrophages in the atherosclerotic plaques. Conclusions—NGAL/24p3 is increased in atherosclerotic plaques and MI. Colocalization with MMP-9 in areas with high-proteolytic activity suggests a role for NGAL/24p3 in modulating the MMP-9-mediated remodeling of plaques and infarcted hearts.

Presence of NGAL/MMP-9 complexes in human abdominal aortic aneurysms

It has been suggested that the intraluminal thrombus of abdominal aortic aneurysms (AAAs) predisposes for AAA enlargement and rupture. The growth of the AAA is dependent on proteolytic degradation of elastin. Here, we analysed whether the neutrophil gelatinase-associated lipocalin (NGAL) is expressed within the thrombus and the aneurysm wall. NGAL can bind to metalloproteinase-9 (MMP-9) and inhibit its degradation, thereby preserving enzymatic activity. Biopsies were obtained from thrombus-free and thrombus-covered aneurysm wall and the intraluminal thrombus from patients undergoing elective surgery for AAA. Immunohistochemistry and real-time PCR were used to study NGAL and MMP-9 expression. Immunoprecipitation, gel zymography, Western blot and ELISA were used to detect and quantify NGAL/MMP-9 complexes. NGAL was detected in the thrombus, the interface between the thrombus and the underlying wall and in the wall itself. Double staining showed that neutrophils are the major source of NGAL expression. Immunoprecipitation of MMP-9 with antibody against NGAL showed that complexes of NGAL and active MMP-9 were present in thrombus, the interface fluid and the aneurysm wall. Western blot analyses using non-reducing conditions and gel zymography demonstrated that high-molecular-weight complexes of NGAL/MMP-9 were present within the different regions. The concentration of the NGAL/MMP-9 complex was highest in the luminal part of the thrombus. In conclusion, NGAL in complex with activated MMP-9 is present in AAA wall and thrombus. Neutrophil-derived NGAL could enhance the proteolytic activity associated with AAA, but the importance of this mechanism for aneurysm growth remains to be shown.

Difference in Matrix-Degrading Protease Expression and Activity Between Thrombus-Free and Thrombus-Covered Wall of Abdominal Aortic Aneurysm

Objective—It has been suggested that the intraluminal thrombus of abdominal aortic aneurysms (AAAs) predisposes for AAA rupture. Here, we examined the possibility that the intraluminal thrombus influences expression and activity of matrix-degrading proteases in the AAA wall. Methods and Results—Twenty patients undergoing elective repair of AAAs were included. From each patient, specimens from both thrombus-covered and thrombus-free wall were taken for analysis. Gene arrays and quantitative real-time polymerase chain reaction showed that matrix metalloproteinase (MMP)-1, -7, -9, and -12 expressions were upregulated in the thrombus-free wall compared with the thrombus-covered wall. Immunohistochemistry confirmed the differential expression of MMP-9 but also localized MMP-9 to the interface between the thrombus and the underlying vessel wall. MMP-9 expression was colocalized with the presence of macrophages. Similar expression patterns were observed for urokinase plasminogen activator (uPA), uPA receptor, and plasminogen activator inhibitor-1. Gelatinase activity was detected in the same regions as MMP-9 protein expression, ie, within the thrombus-free wall and in the interface between the thrombus and the underlying wall. Conclusion—The present work demonstrates that protease expression and activity differs within the aneurysm wall. The source and activity of the proteases responsible for the degradation of the thrombus-covered wall need to be further determined.

Effects of PDGF-C and PDGF-D on monocyte migration and MMP-2 and MMP-9 expression

BACKGROUND AND AIMS Atherosclerosis is a chronic inflammatory process involving the activity of several cytokines and growth factors. Platelet-derived growth factor-A (PDGF-A) and PDGF-B are important mitogens and chemoattractants for monocytes as well as smooth muscle cells. We sought to identify the role of PDGF-C and PDGF-D, two new members of the PDGF family, in monocyte migration and differentiation. We also assessed their effects in regulating matrix metalloproteinase-2 (MMP-2) and MMP-9, which are important for cell migration. METHODS AND RESULTS PDGF-C and PDGF-D were expressed in macrophages, smooth muscle cells, and endothelial cells in human atherosclerotic plaques, as shown by immunohistochemical analysis. PDGF-C and PDGF-D mRNA and protein expression was induced after differentiation of THP-1 monocytes to macrophages, and both PDGF-C and PDGF-D induced MMP-9 mRNA expression in a concentration-dependent manner. Treatment of cells with PDGF-C or PDGF-D enhanced the secretion of MMP-2 and MMP-9 in a cell-dependent manner. In a migration assay using a Boyden chamber with 8 microm pore size, PDGF-C and PDGF-D attracted THP-1 monocytes in a concentration-dependent manner. CONCLUSIONS Our data suggest that PDGF-C and PDGF-D, like PDGF-A and PDGF-B, play important roles in atherosclerosis by stimulating MMP activity and influencing monocyte migration.

Enhanced Cytotoxicity of Nucleoside Analogs by Overexpression of Mitochondrial Deoxyguanosine Kinase in Cancer Cell Lines

The cytotoxic anti-cancer purine nucleoside analogs 2-chloro-2′-deoxyadenosine (CdA), 9-β-d-arabinofuranosylguanine (araG), and 2′,2′-difluorodeoxyguanosine (dFdG) are phosphorylated by human mitochondrial deoxyguanosine kinase (dGK) in vitro. We overexpressed dGK as a fusion protein to the green fluorescent protein in the human pancreatic cancer cell lines PanC-1 and MIA PaCa-2 to determine the importance of dGK-mediated nucleoside analog phosphorylation. The transfected cells showed mitochondrial fluorescence patterns, and the mitochondrial locations of endogenous and overexpressed dGK were verified by Western blot analysis of cell extracts with polyclonal anti-dGK antibodies. The increase of dGK activity in the overexpressing cells was ∼4-fold. These cell lines exhibited increased sensitivity to CdA, araG, and dFdG as compared with the untransfected parent cell lines. This is, to our knowledge, the first demonstration of a correlation between the activity of a mitochondrial deoxyribonucleoside kinase and the cytotoxicity of nucleoside analogs. Our data imply that the dGK activity is rate-limiting for the efficacy of nucleoside analogs in the cell lines investigated.

MMP-2 and MMP-9 are prominent matrix metalloproteinases during atherosclerosis development in the Ldlr-/-Apob100/100 mouse

Matrix-degrading proteases capable of degrading components of the extracellular matrix may play an important role in development and progression of atherosclerotic lesions. In the present study, we used the Ldlr(-/-)Apob(100/100) mouse model, which has a plasma lipoprotein profile similar to that of humans with atherosclerosis, to study the expression of matrix metalloproteinases (MMPs) during early stages of atherosclerosis development. We analyzed the expression of 11 proteases and three protease inhibitors in 5- to 40-week-old Ldlr(-/-)Apob(100/100) mice. Expression and activity of MMP-2 and MMP-9 was increased in advanced atherosclerotic lesions followed by macrophage infiltration as shown by real-time PCR, gel-based and in situ zymography and immunohistochemistry. Expression of other investigated MMPs did not increase during disease progression. However, the mRNA expression of MMP-8 and MMP-13 was down-regulated, which could explain the relatively high amount of collagen observed in the vessels in this model. In conclusion, low proteolytic expression at early stages of atherogenesis and a limited repertoire of proteolytic enzymes were associated with the progression of atherosclerosis in Ldlr(-/-)Apob(100/100) mice. The study suggests that MMP-2 and MMP-9 are the main proteases involved in atherogenesis in this mouse model.

Differential incorporation of 1‐β‐D‐arabinofuranosylcytosine and 9‐β‐D‐arabinofuranosylguanine into nuclear and mitochondrial DNA

The anti‐leukemic nucleoside analogs 1‐β‐D‐arabinofuranosylcytosine (araC) and 9‐β‐D‐arabinofuranosylguanine (araG) are dependent on intracellular phosphorylation for pharmacological activity. AraC is efficiently phosphorylated by deoxycytidine kinase (dCK). Although araG is phosphorylated by dCK in vitro, it is a preferred substrate of mitochondrial deoxyguanosine kinase. We have used autoradiography to show that araC was incorporated into nuclear DNA in Molt‐4 and CEM T‐lymphoblastoid cells as well as in Chinese hamster ovary cells. In contrast, araG was predominantly incorporated into mitochondrial DNA in the investigated cell lines, without detectable incorporation into nuclear DNA. These data suggest that the molecular targets of araG and araC may differ.

Cloning of mouse mitochondrial thymidine kinase 2 cDNA

Phosphorylation of anti‐viral nucleoside analogs by mitochondrial thymidine kinase 2 (TK2) has been implicated as a mechanism for the mitochondrial toxicity caused by several of these compounds. We have cloned the cDNA of mouse TK2 and shown that the enzyme is targeted to the mitochondria when expressed in fusion with the green fluorescent protein. This is the first report on the cloning of a mitochondrial TK2 and will contribute to elucidate the role of TK2 in the pharmacological activation of nucleoside analogs.