CD13 is essential for inflammatory trafficking and infarct healing following permanent coronary artery occlusion in mice.

AIMS: To determine the role of CD13 as an adhesion molecule in trafficking of inflammatory cells to the site of injury in vivo and its function in wound healing following myocardial infarction induced by permanent coronary artery occlusion. METHODS AND RESULTS: Seven days post-permanent ligation, hearts from CD13 knockout (CD13(KO)) mice showed significant reductions in cardiac function, suggesting impaired healing in the absence of CD13. Mechanistically, CD13(KO) infarcts showed an increase in small, endothelial-lined luminal structures, but no increase in perfusion, arguing against an angiogenic defect in the absence of CD13. Cardiac myocytes of CD13(KO) mice showed normal basal contractile function, eliminating myocyte dysfunction as a mechanism of adverse remodelling. Conversely, immunohistochemical and flow cytometric analysis of CD13(KO) infarcts demonstrated a dramatic 65% reduction in infiltrating haematopoietic cells, including monocytes, macrophages, dendritic, and T cells, suggesting a critical role for CD13 adhesion in inflammatory trafficking. Accordingly, CD13(KO) infarcts also contained fewer myofibroblasts, consistent with attenuation of fibroblast differentiation resulting from the reduced inflammation, leading to adverse remodelling. CONCLUSION: In the ischaemic heart, while compensatory mechanisms apparently relieve potential angiogenic defects, CD13 is essential for proper trafficking of the inflammatory cells necessary to prime and sustain the reparative response, thus promoting optimal post-infarction healing.

CD13 is dispensable for normal hematopoiesis and myeloid cell functions in the mouse.

The robust and consistent expression of the CD13 cell surface marker on very early as well as differentiated myeloid hematopoietic cells has prompted numerous investigations seeking to define roles for CD13 in myeloid cells. To address the function of myeloid CD13 directly, we created a CD13 null mouse and assessed the responses of purified primary macrophages or DCs from WT and CD13 null animals in cell assays and inflammatory disease models, where CD13 has been implicated previously. We find that mice lacking CD13 develop normally with normal hematopoietic profiles except for an increase in thymic but not peripheral T cell numbers. Moreover, in in vitro assays, CD13 appears to be largely dispensable for the aspects of phagocytosis, proliferation, and antigen presentation that we tested, although we observed a slight decrease in actin-independent erythrocyte uptake. However, in agreement with our published studies, we show that lack of monocytic CD13 completely ablates anti-CD13-dependent monocyte adhesion to WT endothelial cells. In vivo assessment of four inflammatory disease models showed that lack of CD13 has little effect on disease onset or progression. Nominal alterations in gene expression levels between CD13 WT and null macrophages argue against compensatory mechanisms. Therefore, although CD13 is highly expressed on myeloid cells and is a reliable marker of the myeloid lineage of normal and leukemic cells, it is not a critical regulator of hematopoietic development, hemostasis, or myeloid cell function.

CD13/APN transcription is regulated by the proto-oncogene c-Maf via an atypical response element.

Angiogenic growth factors induce the transcription of the cell surface peptidase CD13/APN in activated endothelial cells of the tumor vasculature. Inhibition of CD13/APN abrogates endothelial invasion and morphogenesis in vitro and tumor growth in vivo suggesting a critical functional role for CD13 in angiogenesis. Experiments to identify the transcription factors responsible for this regulation demonstrated that exogenous expression of the proto-oncogene c-Maf, but not other bZip family members tested, potently activates transcription from a critical regulatory region of the CD13 proximal promoter between -115 and -70 bp which is highly conserved among mammalian species. Using promoter mutation, EMSA and ChIP analyses we established that both endogenous and recombinant c-Maf directly interact with an atypical Maf response element contained within this active promoter region via its basic DNA/leucine zipper domain. However full activity of c-Maf requires the amino-terminal transactivation domain, and site-directed mutation of putative phosphorylation sites within the transactivation domain (serines 15 and 70) shows that these sites behave in a dramatic cell type-specific manner. Therefore, this atypical response element predicts a broader range of c-Maf target genes than previously appreciated and thus impacts its regulation of multiple myeloma as well as endothelial cell function and angiogenesis.

Expression of ubiquitin carboxy-terminal hydrolase-l1 in photocoagulated human retinal pigment epithelial cells.

PURPOSE: To date, the exact mechanisms involved in laser-induced remission of ocular neovascular disorders are still poorly understood. Recent studies suggest that the expression of various antiangiogenic cytokines is upregulated after thermal photocoagulation. In the current study, we sought to identify novel laser-regulated proteins in cultured human retinal pigment epithelial (HRPE) cells. METHODS: Protein extracts from photocoagulated HRPE cells were subjected to 2D-gel electrophoresis. Potentially regulated protein spots were identified by mass spectroscopy. Regulation of protein and mRNA was determined by Western blot analysis and reverse transcription-polymerase chain reaction, respectively. RESULTS: 2D-Gel electrophoresis of HRPE whole-cell extracts exposed to photocoagulation revealed a reproducible increase in a protein band identified as ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) compared with untreated controls. Protein levels showed a time-dependent upregulation over 24 hr. UCH-L1 mRNA was maximally increased after 8 hr. CONCLUSIONS: Our findings indicate that the ubiquitin-proteasome system contributes to the effects seen clinically after thermal photocoagulation in eyes with neovascular diseases of the retina or choroid. Because ubiquitin carboxy-terminal hydrolase-L1 has been shown to be involved in the regulation of cell cycle proteins, it may be speculated that deubiquitinating enzymes have a role in the regeneration and proliferation of retinal pigment epithelial cells.

CD13/APN regulates endothelial invasion and filopodia formation.

CD13/aminopeptidase N is a transmembrane peptidase that is induced in the vasculature of solid tumors and is a potent angiogenic regulator. Here, we demonstrate that CD13 controls endothelial cell invasion in response to the serum peptide bradykinin by facilitating signal transduction at the level of the plasma membrane. Inhibition of CD13 abrogates bradykinin B(2) receptor internalization, leading to the attenuation of downstream events such as bradykinin-induced activation of Cdc42 and filopodia formation, and thus affects endothelial cell motility. Investigation into mechanisms underlying this block led us to focus on B(2)R internalization via membrane-dependent mechanisms. Membrane disruption by depletion of cholesterol or trypsinization halts B(2)R internalization, invasion, and filopodia formation, which can be recovered with addition of cholesterol. However, this functional recovery is severely impaired in the presence of CD13 antagonists, and the distribution of membrane proteins is disordered in treated cells, suggesting a role for CD13 in plasma membrane protein organization. Finally, exogenous expression of wild-type but not mutant CD13 further alters protein distribution, suggesting peptidase activity is required for CD13's regulatory activity. Therefore, CD13 functions as a novel modulator of signal transduction and cell motility via its influence on specific plasma membrane organization, thus regulating angiogenesis.

Pigment-epithelium-derived factor is upregulated in photocoagulated human retinal pigment epithelial cells.

There is much evidence that pigment-epithelium-derived factor (PEDF) is a potent antiangiogenic cytokine which inhibits retinal and choroidal neovascularization by inducing apoptosis in activated vascular endothelial cells. Furthermore, the regulation of PEDF appears to be linked to the regulation of vascular endothelial growth factor (VEGF), one of the most potent inducers of intraocular neovascularization. Previous studies have established that thermal photocoagulation, the mainstay in the therapy of various neovascular diseases of the posterior segment, results in a decrease in intraocular concentrations of VEGF and other angiogenic growth factors, thereby inhibiting active retinal neovascularization. In the current study, we sought to determine whether thermal photocoagulation has the potential to regulate the expression of PEDF in human retinal pigment epithelial (RPE) cells. Cultures of RPE cells were photocoagulated with a 532-nm diode laser. Subsequently, RNA was isolated for RT-PCR, and whole-cell extracts and precipitated cell culture supernatant were subjected to Western blot analysis. According to our results, PEDF mRNA and protein are significantly upregulated after photocoagulation. Moreover, PEDF protein was found to be secreted in the cell culture medium.

Modulation of tissue plasminogen activator and plasminogen activator inhibitor-1 by transforming growth factor-beta in human retinal glial cells.

PURPOSE: The serine proteases tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) and their inhibitor, plasminogen activator inhibitor (PAI)-1, regulate a variety of processes involved in tissue morphogenesis and differentiation. There is much evidence that plasminogen activator-mediated extracellular matrix degradation is an important step in the development of ocular neovascular diseases. The authors investigated whether expression of t-PA, u-PA, and PAI-1 in human retinal glial cells (HRGCs) is influenced by exposure to transforming growth factor (TGF)-beta, a cytokine that regulates the proliferation and differentiation of cells. METHODS: The extracellular release of t-PA, u-PA, and PAI-1 was measured by enzyme-linked immunosorbent assay (ELISA) in the supernatant of HRGC cultures, under basal conditions and after stimulation with TGF-beta at various concentrations (2, 5, 10, or 20 ng/mL). Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze mRNA levels. Smad2 phosphorylation was detected by Western blot analysis. RESULTS: Under basal conditions, HRGCs secreted considerable amounts of t-PA and PAI-1. Stimulation with TGF-beta resulted in increased synthesis of t-PA and PAI-1 protein in a time- and dose-dependent manner. Moreover, an increased expression of t-PA and PAI-1 mRNA after supplementation with TGF-beta was observed, with maximum expression at 12 hours. In contrast, HRGCs did not respond to TGF-beta with any change of u-PA production, although there were detectable amounts of u-PA mRNA and protein. Phosphorylation of Smad2 was increased after addition of TGF-beta. This effect was partially reversible after treatment with interferon-gamma. CONCLUSIONS: The production of plasminogen activators and PAI-1 by HRGCs reflects the potential role of these cells in the progression of neovascular ocular diseases. Furthermore, the finding that t-PA and PAI-1 synthesis by HRGCs is mediated by TGF-beta and its downstream effector Smad2 confirms the importance of the TGF-beta signaling pathway in the regulation of interactions between retinal cells and the extracellular matrix.