Michael Pech

Connective tissue growth factor (CTGF) stimulates vascular smooth muscle cell growth and migration in vitro

Connective tissue growth factor (CTGF) was first identified as a 38-kDa cysteine-rich protein which can be specifically induced by TGF-beta and was recently found to be expressed abundantly in atherosclerotic lesions, but only marginally in normal vascular tissues. It was hypothesized that CTGF is one of the factors involved in the development of atherosclerotic lesions. In this study, we investigated the functions of CTGF protein in regulating the growth and migration of vascular smooth muscle cells (VSMC) and found that by overexpressing CTGF in VSMC, proliferation and migration rates were significantly increased. The accelerated growth and migration can be reversed by an anti-CTGF antibody. In addition, overexpression of CTGF also promotes VSMC to express more extracellular matrix protein collagen I and fibronectin. Our results indicate that CTGF is a growth factor for VSMC and it may play a similar role in promoting VSMC proliferation, migration, and formation of extracellular matrix, in vivo.

ADP-ribosylation factor (ARF)-like 7 (ARL7) is induced by cholesterol loading and participates in apolipoprotein AI-dependent cholesterol export

Here, we identify ADP‐ribosylation factor (ARF)‐like 7 (ARL7) as the only ARF‐ and ARL‐family member whose mRNA‐expression is induced by liver X‐receptor/retinoid X‐receptor agonists or cholesterol loading in human macrophages. Moreover, subcellular distribution of mutant and wild type ARL7‐enhanced green fluorescent protein (EGFP) supports that ARL7 may be involved in a vesicular transport step between a perinuclear compartment and the plasma membrane. Therefore, we investigated the effect of ARL7 over‐expression on the cholesterol secretory pathway. We found that expression of wild type and dominant active ARL7‐EGFP stimulated the rate of apolipoprotein AI‐specific cholesterol efflux 1.7‐ and 2.8‐fold. In contrast, expression of the dominant negative form of ARL7‐EGFP led to ∼50% inhibition of cholesterol efflux. This data is consistent with a model in which ARL7 is involved in transport between a perinuclear compartment and the plasma membrane apparently linked to the ABCA1‐mediated cholesterol secretion pathway.

Connective tissue growth factor in retrocorneal membranes and corneal scars.

We studied the localization and distribution of connective tissue growth factor (CTGF) in corneal scar tissue and membranes using in situ hybridization in 8 corneas from keratoplasty and 4 normal corneas. Identification of the cells was done with immunohistochemistry for SM-α-actin, vimentin, and Lu5. CTGF mRNA was found in activated corneal fibroblasts in 7 of 8 scars, 7 of 8 retrocorneal membranes and 2 subepithelial membranes, whereas the control corneas showed no CTGF mRNA expression. Vimentin was positive in all scars, retrocorneal and subepithelial membranes, SM-α-actin in 7 of 8 scars and 6 of 8 retrocorneal membranes. These results suggest that CTGF plays a crucial role in corneal wound healing and membrane formation.

Mechanism of Inhibition of Neointimal Formation by the Angiotensin-Converting Enzyme Inhibitor Cilazapril A Study in Balloon Catheter–Injured Rat Carotid Arteries

We investigated the mechanism of inhibition of neointima formation by the angiotensin-covering enzyme the carotid artery. We looked for the effects of cilazapril on all phases of the response to injury, ie, on proliferation of smooth muscle cells (SMCs) in the media, their migration, their proliferation in the neointima, and their disposition of extracellular matrix in the neointima. Although treatment was discontinued after 2 weeks, the inhibitory effect of cilazapril on neointimal formation was evident even 52 weeks after injury. The amount of extracellular matrix deposited in the intima during cilazapril treatment was decreased by 20% 2 weeks after injury, but no effect was seen if tissues were analyzed at 4 or 52 weeks. [3H]Thymidine-labeled cells (pulse labeling as well as 14-day continuous labeling) showed a decrease in SMC labeling in the tunica medica by 50%, but no inhibition in the labeling indices was seen in the neointima. The fraction of unlabeled neointimal cells in the cilazapril-treated rats as judged from continuous labeling experiments was inhibited by 86%. Taken together, these data suggest an antiproliferative effect on medial SMCs and an inhibition of SMC migration into the intima by cilazapril. Since intimal extracellular matrix deposition was only delayed, the decrease in medial SMC proliferation and subsequent migration seems to be the main reason for the reduction of neointima formation.

Conservation in sequence and affinity of human and rodent PDGF ligands and receptors

Platelet-derived growth factor (PDGF) consists of two chains, PDGF-A and -B, which activate as homo- or heterodimers two receptors, alpha and beta. To test PDGF function in vivo we have generated neutralizing monoclonal antibodies. When analyzed with rat PDGFs only antibodies raised against human PDGF-AA showed cross-species activity. This correlated with complete amino acid sequence conservation of PDGF-A whereas rat PDGF-B differed in six positions when cloned rat PDGF cDNAs were compared with their human homologs within the receptor binding region. Extracellular domains of cloned rat PDGF alpha- and beta-receptor cDNAs did not reflect this difference in cross-species ligand conservation. When rat extracellular domains were expressed as soluble proteins they bound human PDGF-BB with high affinity after immobilization of the purified proteins on solid phase. Dissociation constants were identical to those of their human homologs. Thus, high affinity binding of human PDGF-BB to extracellular domains does not depend on species origin but only on receptor type.

Trimerized apolipoprotein A-I (TripA) forms lipoproteins, activates lecithin: cholesterol acyltransferase, elicits lipid efflux, and is transported through aortic endothelial cells.

Apolipoprotein A-I (apoA-I) exerts many potentially anti-atherogenic properties and is therefore attractive for prevention and therapy of coronary heart disease. Since induction of apoA-I production by small molecules has turned out as difficult, application of exogenous apoA-I is pursued as an alternative therapeutic option. To counteract fast renal filtration of apoA-I, a trimeric high-molecular weight variant of apoA-I (TripA) was produced by recombinant technology. We compared TripA and apoA-I for important properties in reverse cholesterol transport. Reconstituted high-density lipoproteins (rHDL) containing TripA or apoA-I together with palmitoyl-2-oleyl-phosphatidylcholine (POPC) differed slightly by size. Compared to apoA-I, TripA activated lecithin:cholesterol acyltransferase (LCAT) with similar maximal velocity but concentration leading to half maximal velocity was slightly reduced (K(m)=2.1±0.3μg/mL vs. 0.59±0.06μg/mL). Both in the lipid-free form and as part of rHDL, TripA elicited cholesterol efflux from THP1-derived macrophages with similar kinetic parameters and response to liver-X-receptor activation as apoA-I. Lipid-free TripA is bound and transported by aortic endothelial cells through mechanisms which are competed by apoA-I and TripA and inhibited by knock-down of ATP-binding cassette transporter (ABC) A1. Pre-formed TripA/POPC particles were bound and transported by endothelial cells through mechanisms which are competed by excess native HDL as well as reconstituted HDL containing either apoA-I or TripA and which involve ABCG1 and scavenger receptor B1 (SR-BI). In conclusion, apoA-I and TripA show similar in vitro properties which are important for reverse cholesterol transport. These findings are important for further development of TripA as an anti-atherosclerotic drug.

Expression of a Rat PDGF Receptor β Ectodomain Generates a Low Affinity Ligand Antagonist

Platelet-derived growth factor (PDGF) controls migration and proliferation of mesenchymal cells and is thought to be involved in the pathophysiology of different diseases such as arteriosclerosis and tumorigenesis. In order to investigate the role of PDGF in rat models for such diseases, sufficient amounts of PDGF antagonists are needed. For this purpose we expressed the extracellular domain (ectodomain) of the rat PDGF receptor beta (PDGFR beta) in insect cells using a baculovirus vector. A hydrophilic octapeptide was added onto the N-terminus of the receptor ectodomain to follow its expression with specific anti-FLAG antibodies. The FLAG tag was also utilized to design a rapid two-step purification protocol. Purified FLAG-tagged rat PDGFR beta ectodomain had an affinity to PDGF-BB identical to the untagged ectodomain as determined by Scatchard analysis. FLAG-tagged PDGFR beta ectodomain in solution, however, did not compete for PDGF-BB binding to full length cellular receptors at concentrations expected for an high affinity antagonist.