Safety of a non-viral plasmid-encoding dual isoforms of hepatocyte growth factor in critical limb ischemia patients: a phase I study.

We aimed to evaluate in a phase I dose-escalation study, the safety of intramuscular injections of a novel non-viral plasmid DNA expressing two isoforms of human hepatocyte growth factor (HGF) (VM202) in patients with critical limb ischemia (CLI). In total, 12 patients with CLI and unsuitable for revascularization were consecutively assigned to increasing doses (2 to 16 mg) of VM202 administered into the ischemic calf muscle at days 1 and 15. Patients were evaluated for safety and tolerability, changes in ankle- and toe brachial index (ABI and TBI), and pain severity score using a visual analog scale (VAS) throughout a 12-month follow-up period. Median age was 72 years and 53% of the patients were male. VM202 was safe and well tolerated with no death during the 12-month follow-up. Median ABI and TBI significantly increased from 0.35 to 0.52 (P=0.005) and from 0.15 to 0.24 (P=0.01) at 12 months follow-up. Median VAS decreased from 57.5 to 16.0 mm at 6 months follow-up (P=0.03). In this first human clinical trial, VM202, which expresses two isoforms of human HGF, appear to be safe and well tolerated with encouraging clinical results and thus supports the performance of a phase II randomized controlled trial.

Catheter-based methods for managing peripheral vascular disease. Which types of occlusive disease are most amenable?

Primary care physicians have an important role in identification and referral of patients with signs and symptoms of peripheral vascular disease. Advances in technology have increased the effectiveness of percutaneous catheter-based techniques used to treat stenosis or occlusion in various anatomic sites. However, early evaluation remains an important factor in the success of treatment. In this symposium article, Drs Lips and Vacek review the current catheter-based techniques for managing various types of occlusive disease and include numerous angiographic illustrations from their case files. They also examine the factors affecting choice of treatment approach and briefly discuss the role of thrombolytic therapy.

The discovery of a 3-phosphomonoesterase that hydrolyzes phosphatidylinositol 3-phosphate in NIH 3T3 cells.

Phosphatidylinositol 3-phosphate (PtdIns(3)P), a recently described phospholipid, has been linked to polyoma virus-induced cellular transformation and platelet-derived growth factor-mediated mitogenesis. PtdIns(3)P, in contrast to phosphatidylinositol, phosphatidylinositol 4-phosphate (PtdIns(4)P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), is resistant to hydrolysis by bovine brain phospholipase C gamma. We present here the identification of a phosphomonoesterase activity from the soluble fraction of NIH 3T3 cells which removes the phosphate from the D-3 position of PtdIns(3)P. This enzyme is specific as it has little or no activity on the monoester phosphates of PtdIns(4)P, PtdIns(4,5)P2, or inositol 1,3-bisphosphate and is tentatively designated phosphatidylinositol 3-phosphatase (PtdIns 3-phosphatase). The enzyme does not require added metal ions for activity and is maximally active in the presence of EDTA. It is inhibited by Ca2+, Mg2+, Zn2+, and the phosphatase inhibitor VO4(3-). In addition, there is no phospholipase C activity toward PtdIns(3)P in the soluble fraction of NIH 3T3 cells. In view of the absence of a phospholipase C activity that hydrolyzes PtdIns(3)P, we propose that PtdIns(3)P is not a precursor for a soluble inositol phosphate messenger but that it instead may act directly to control certain cellular processes or as a precursor for other phosphatidylinositols. PtdIns 3-phosphatase may thus terminate a metabolic signal or regulate precursor levels for other phosphatidylinositols that are phosphorylated in the D-3 position.

Isolation of 1-monomethylphosphoinositol 4,5-bisphosphate [a product of methanolysis of inositol 1,2-(cyclic)-4,5-trisphosphate] from Swiss mouse 3T3 cells.

We have noted two previously undescribed inositol polyphosphates in neutral methanol extracts from Swiss mouse 3T3 cells that were grown in [3H]inositol and stimulated with platelet-derived growth factor. They have been identified as 1-monomethylphosphoinositol 4,5-bisphosphate and 1-monomethylphosphoinositol 4-phosphate by comparison to a synthesized standard using HPLC chromatography, paper electrophoresis, and enzymatic dephosphorylation with inositol polyphosphate 5-phosphomonoesterase and intestinal alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1]. We propose that these compounds are formed by methanolysis of inositol 1,2-(cyclic)-4,5-trisphosphate and inositol 1,2-(cyclic)-4-bisphosphate present in the cells. Inositol cyclic phosphates did not react with neutral methanol in the absence of the cells, which are required for the methanolysis reaction. These findings suggest a role for inositol cyclic phosphates as reactive compounds that are added to as yet unidentified cellular acceptors.

Isolation and characterization of two 3-phosphatases that hydrolyze both phosphatidylinositol 3-phosphate and inositol 1,3-bisphosphate.

Inositol-polyphosphate 3-phosphatase catalyzes the hydrolysis of the 3-position phosphate bond of inositol 1,3-bisphosphate (Ins(1,3)P2) to form inositol 1-monophosphate and inorganic phosphate (Bansal, V.S., Inhorn, R.C., and Majerus, P.W. (1987) J. Biol. Chem. 262, 9444-9447). Phosphatidylinositol 3-phosphatase catalyzes the analogous reaction utilizing phosphatidylinositol 3-phosphate (PtdIns(3)P) as substrate to form phosphatidylinositol and inorganic phosphate (Lips, D.L., and Majerus, P.W. (1989) J. Biol. Chem. 264, 19911-19915). We now demonstrate that these enzyme activities are identical. Two forms of the enzyme, designated Type I and II 3-phosphatases, were isolated from rat brain. The Type I 3-phosphatase consisted of a protein doublet that migrated at a relative Mr of 65,000 upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The Mr of this isoform upon size-exclusion chromatography was 110,000, suggesting that the native enzyme is a dimer. The Type II enzyme consisted of equal amounts of an Mr = 65,000 doublet and an Mr = 78,000 band upon SDS-polyacrylamide gel electrophoresis. This isoform displayed an Mr upon size-exclusion chromatography of 147,000, indicating that it is a heterodimer. The Type II 3-phosphatase catalyzed the hydrolysis of Ins(1,3)P2 with a catalytic efficiency of one-nineteenth of that measured for the Type I enzyme, whereas PtdIns(3)P was hydrolyzed by the Type II 3-phosphatase at three times the rate measured for the Type I 3-phosphatase. The Mr = 65,000 subunits of the two forms of 3-phosphatase appear to be the same based on co-migration on SDS-polyacrylamide gels and peptide maps generated with Staphylococcus aureus protease V8 and trypsin. The peptide map of the Mr = 78,000 subunit was different from that of the Mr = 65,000 subunits. Thus, we propose that the differing relative specificities of the Type I and II 3-phosphatases for Ins(1,3)P2 and PtdIns(3)P are due to the presence of the Mr = 78,000 subunit of the Type II enzyme.

Phosphatidylinositol 3-phosphate is present in normal and transformed fibroblasts and is resistant to hydrolysis by bovine brain phospholipase C II.

The transforming protein of polyoma virus, middle T antigen, associates with two cellular enzymes, pp60c-src, a protein tyrosine kinase, and a phosphatidylinositol kinase that forms phosphatidylinositol 3-phosphate. The formation of a ternary complex of these proteins is essential for complete transformation and maximal tumor induction by the virus. A mutant virus encoding an altered middle T protein that activates pp60c-src but fails to bind phosphatidylinositol kinase is partially defective in transformation. We have confirmed, using an enzymological method, that the product of the in vitro reaction catalyzed by middle T-pp60c-src-phosphatidylinositol kinase complexes is phosphatidylinositol 3-phosphate (PtdIns(3)P), as previously reported (Whitman, M., Downes, C. P., Keeler, M., Keller, T., and Cantley, L. (1988) Nature 332, 644-646). PtdIns(3)P is present in normal as well as virus-infected and transformed cells at levels ranging from 0.6 to 2.6% of the major phosphatidylinositol phosphate isomer, phosphatidylinositol 4-phosphate (PtdIns(4)P). Steady-state levels of PtdIns(3)P do not appear to be affected by the expression of middle T in cells. PtdIns(3)P is not hydrolyzed by bovine brain phospholipase C II, which readily cleaves PtdIns(4)P and other phosphatidylinositols. This result underscores the likelihood that the metabolism of PtdIns(3)P is distinct from that of PtdIns(4)P and raises further questions regarding a possible role of PtdIns(3)P in normal and neoplastic cell growth.

Pathway for the formation of D-3 phosphate containing inositol phospholipids in intact human platelets.

We have identified the structure of phosphatidylinositol 3-phosphate (PtdIns(3)P), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in human platelets. These lipids accounted for less than 2% of the total 32P incorporated into inositol phospholipids in the platelets. All three lipids were labeled in unstimulated platelets, but incorporation of 32P changed rapidly by 15 s after thrombin stimulation, suggesting that they are important in platelet activation. Specific inositol polyphosphate phosphatases were used to both identify the lipid structures and to determine the route of synthesis of these lipids. During 32P labeling and after thrombin stimulation of human platelets, as much as 60% of the total radioactivity present in PtdIns(3,4)P2 was found in the D-4 phosphate and only 35% in the D-3 phosphate indicating that PtdIns(3)P is the precursor of PtdIns(3,4)P2. In addition, the D-5 and D-4 phosphates of PtdIns(3,4,5)P3 each contained 35-40% of the total radioactivity in the molecule compared with only 18-28% in the D-3 position, suggesting that PtdIns(3,4)P2 and not PtdIns(4,5)P2 is the major precursor of this lipid. These results define the predominant pathway for synthesis of these lipids in platelets as PtdIns----PtdIns(3)P----PtdIns(3,4)P2----PtdIns(3,4,5)P3.

Radiation exposure: comparison of rapid exchange and conventional over-the-wire coronary angioplasty systems.

The growth of coronary angioplasty has resulted in increased fluoroscopy time to patients, staff, and physicians. Rapid exchange-type catheters have purported to reduce fluoroscopy time and procedure time compared to conventional over-the-wire systems. Of 150 consecutive patients, 54 were treated solely with rapid-exchange catheters and 84 were treated solely with over-the-wire catheters. Excluding 12 cases treated with fixed wire or combination catheters, the following data were found: [table: see text] Overall procedural success was obtained in 98.5% (136/138) of patients, 1.5% (2) requiring emergency coronary artery bypass graft surgery. A significant 35% reduction in fluoroscopy time and 13% reduction in procedure time were found when rapid-exchange catheters were used, with identical success rates, number of balloons used per case, and complication rates compared to over-the-wire catheters. Rapid-exchange catheters should be considered as part of an overall effort to reduce radiation exposure in the interventional cardiology laboratory.