A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter.

Picolinic acid, a catabolite of L-tryptophan, activates the transcription of the inducible nitric oxide synthase gene (iNOS) in IFN-gamma-treated murine macrophages. We performed functional studies on the 5' flanking region of the iNOS gene linked to a CAT reporter gene to identify the cis-acting element(s) responsible for the activation of iNOS transcription by picolinic acid. Transient transfection assays showed that the full-length iNOS promoter in the murine macrophage cell line ANA-1 was activated by the synergistic interaction between IFN-gamma and picolinic acid. Deletion or mutation of the iNOS promoter region from -227 to -209, containing a sequence homology to a hypoxia-responsive enhancer (iNOS-HRE), decreased picolinic acid- but not LPS-induced CAT activity by more than 70%. Functional studies using a tk promoter-CAT reporter gene plasmid demonstrated that the iNOS-HRE was sufficient to confer inducibility by picolinic acid but not by IFN-gamma or LPS. Electrophoretic mobility shift assays confirmed that picolinic acid alone induced a specific binding activity to the iNOS-HRE. Furthermore, we found that the iNOS-HRE activity was inducible by hypoxia and that hypoxia in combination with IFN-gamma activated the iNOS promoter in transient transfection assays and induced iNOS transcription and mRNA expression. These data establish that the iNOS-HRE is a novel regulatory element of the iNOS promoter activity in murine macrophages and provide the first evidence that iNOS is a hypoxia-inducible gene.

The Oxidative Stress-Induced miR-200c Is Upregulated in Psoriasis and Correlates with Disease Severity and Determinants of Cardiovascular Risk.

Psoriasis is a chronic inflammatory skin disease associated with reactive oxygen species (ROS) increase and a higher risk of cardiovascular (CV) events. We previously showed that the miR-200 family (miR-200s) is induced by ROS, miR-200c being the most upregulated member responsible for apoptosis, senescence, ROS increase, and nitric oxide decrease, finally causing endothelial dysfunction. Moreover, circulating miR-200c increases in familial hypercholesterolemic children and in plaques and plasma of atherosclerotic patients, two pathologies associated with increased ROS. Given miR-200s' role in endothelial dysfunction, ROS, and inflammation, we hypothesized that miR-200s were modulated in lesional skin (LS) and plasma of psoriatic patients (Pso) and that their levels correlated with some CV risk determinants at a subclinical level. All Pso had severe psoriasis, i.e., Psoriasis Area and Severity Index (PASI) > 10, and one of the following: at least two systemic psoriasis treatments, age at onset < 40 years, and disease duration > 10 years. RNA was extracted from plasma (Pso, N = 29; Ctrl, N = 29) and from nonlesional skin (NLS) and LS of 6 Pso and 6 healthy subject skin (HS) biopsies. miR-200 levels were assayed by quantitative RT-PCR. We found that all miR-200s were increased in LS vs. NLS and miR-200c was the most expressed and upregulated in LS vs. HS. In addition, circulating miR-200c and miR-200a were upregulated in Pso vs. Ctrl. Further, miR-200c positively correlated with PASI, disease duration, left ventricular (LV) mass, LV relative wall thickness (RWT), and E/e', a marker of diastolic dysfunction. Multiple regression analysis indicates a direct association between miR-200c and both RWT and LV mass. Circulating miR-200a correlated positively only with LV mass and arterial pressure augmentation index, a measure of stiffness, although the correlations were nearly significant (P = 0.06). In conclusion, miR-200c is upregulated in LS and plasma of Pso, suggesting its role in ROS increase and inflammation associated with CV risk in psoriasis.

UVC inhibits HIF-1alpha protein translation by a DNA damage- and topoisomerase I-independent pathway.

Hypoxia inducible factor 1 (HIF-1) is a key player in cancer progression and an attractive target for cancer therapy. Several small molecule inhibitors of HIF-1alpha also induce a DNA damage response. However, whether or not DNA damage is required for or associated with the inhibition of HIF-1alpha protein accumulation is poorly understood. In this report we investigated the effects of distinct DNA damaging conditions on the hypoxic induction of HIF-1alpha protein in cancer cell lines. We demonstrate that in addition to topotecan (TPT), a known inhibitor of HIF-1alpha, UVC, but not other DNA damaging agents (cisplatin, ionizing radiation and doxorubicin), inhibited HIF-1alpha protein accumulation in a dose-dependent, p53-independent fashion. Low doses UVC decreased HIF-1alpha translation without affecting global protein synthesis. Inhibition of HIF-1alpha by UVC required ongoing RNA transcription, but not DNA replication. Moreover, a functional ATR was required for the activation of DNA damage-dependent responses by both UVC and TPT, but was dispensable for the inhibition of HIF-1alpha protein. Notably, unlike TPT, inhibition of HIF-1alpha protein by UVC did not require topoisomerase I, suggesting a similar yet distinct mode of action. Our data reveal that UVC is a novel signal associated with inhibition of HIF-1alpha protein accumulation, and they uncouple the DNA damage-dependent signaling pathway exerted by UVC and TPT from HIF-1alpha inhibition.

Intracellular signaling pathways required for rat vascular smooth muscle cell migration. Interactions between basic fibroblast growth factor and platelet-derived growth factor.

Intracellular signaling pathways activated by both PDGF and basic fibroblast growth factor (bFGF) have been implicated in the migration of vascular smooth muscle cells (VSMC), a key step in the pathogenesis of many vascular diseases. We demonstrate here that, while bFGF is a weak chemoattractant for VSMCs, it is required for the PDGF-directed migration of VSMCs and the activation of calcium/calmodulin-dependent protein kinase II (CamKinase II), an intracellular event that we have previously shown to be important in the regulation of VSMC migration. Neutralizing antibodies to bFGF caused a dramatic reduction in the size of the intracellular calcium transient normally seen after PDGF stimulation and inhibited both PDGF-directed VSMC migration and CamKinase II activation. Partially restoring the calcium transient with ionomycin restored migration and CamKinase II activation as did the forced expression of a mutant CamKinase II that had been "locked" in the active state by site-directed mutagenesis. These results suggest that bFGF links PDGF receptor stimulation to changes in intracellular calcium and CamKinase II activation, reinforcing the central role played by CamKinase II in regulating VSMC migration.

Acidosis inhibits endothelial cell apoptosis and function and induces basic fibroblast growth factor and vascular endothelial growth factor expression.

Endothelial cells are exposed to an acidotic environment in a variety of pathological and physiological conditions. However, the effect of acidosis on endothelial cell function is still largely unknown, and it was evaluated in the present study. Bovine aortic endothelial cells (BAECs) were grown in bicarbonate buffer equilibrated either with 20% CO(2) (pH 7.0, acidosis) or 5% CO(2) (pH 7.4, control). Acidosis inhibited BAEC proliferation in 10% FCS, whereas by day 7 in serum-free medium, cell number was 3-fold higher in acidotic cells than in control cells. Serum deprivation enhanced BAEC apoptosis, and apoptotic cell death was markedly inhibited by acidosis. Additionally, acidosis inhibited FCS-stimulated migration in a modified Boyden chamber assay and FCS-stimulated differentiation into capillary-like structures on reconstituted basement membrane proteins. Conditioned media from BAECs cultured for 48 hours either at pH 7.0 or pH 7.4 enhanced BAEC proliferation and migration at pH 7.4, and both effects were more marked with conditioned medium from BAECs grown in acidotic than in control conditions. Acidosis enhanced vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) mRNA expression as well as bFGF secretion, and a blocking bFGF antibody inhibited enhanced BAEC migration in response to conditioned medium from acidotic cells. These results show that acidosis protects endothelial cells from apoptosis and inhibits their proangiogenic behavior despite enhanced VEGF and bFGF mRNA expression and bFGF secretion.

Bryostatin-1 and IFN-gamma synergize for the expression of the inducible nitric oxide synthase gene and for nitric oxide production in murine macrophages.

Bryostatin-1 (Bryo) is a nontumor-promoting protein kinase C modulator that has been shown to have both in vitro and in vivo activity against several murine and human tumors. In this study, we investigated the effects of Bryo on nitric oxide production, measured as accumulated nitrite (NO2-) in culture supernatant, and inducible nitric oxide synthase (iNOS) gene expression in the murine macrophage cell line ANA-1. ANA-1 macrophages did not produce NO2- or iNOS mRNA constitutively, and very little or no NO2- or iNOS mRNA were detectable upon exposure to IFN-gamma. Bryo, although ineffective alone, and IFN-gamma synergized to produce high levels of NO2- and iNOS mRNA. The activity of Bryo was evident at a concentration of 0.1 ng/ml and reached its maximum at 1 ng/ml. The effects of Bryo were time dependent because expression of iNOS mRNA was detectable as early as 6 h and increased through 24 h. Analyses of the molecular mechanisms involved indicate that Bryo and IFN-gamma mainly regulate iNOS gene expression posttranscriptionally through stabilization of iNOS mRNA. Experiments designed to investigate the role of tumor necrosis factor alpha (TNF-alpha) in NO2- production by Bryo- and IFN-gamma-activated macrophages revealed that ANA-1 macrophages expressed low levels of TNF-alpha mRNA constitutively that were not augmented in the presence of IFN-gamma. However, Bryo alone augmented the TNF-alpha mRNA expression, which was only slightly increased with the addition of IFN-gamma. A polyclonal antibody to TNF-alpha was able to completely neutralize TNF-alpha secreted in either medium or Bryo plus IFN-gamma-treated cultures. Neutralizing concentrations of anti-TNF-alpha antibody suppressed the Bryo plus IFN-gamma-induced NO2- production approximately by 50%, suggesting that NO2- produced by Bryo plus IFN-gamma-treated ANA-1 macrophages may involve both TNF-alpha-dependent and TNF-alpha-independent mechanisms. Overall, these findings provide the first evidence that Bryo and IFN-gamma can synergize for the induction of NO2- production as well as iNOS gene expression and show the involvement of posttranscriptional mechanisms in the induction of iNOS mRNA.

Flavopiridol, a protein kinase inhibitor, down-regulates hypoxic induction of vascular endothelial growth factor expression in human monocytes.

We have investigated the effects of flavopiridol, a novel protein kinase inhibitor that is selective for cyclin-dependent kinases, on hypoxia-induced vascular endothelial growth factor (VEGF) expression in human monocytes. We found that hypoxia induces a time-dependent increase of VEGF mRNA expression and protein levels in human monocytes. Flavopiridol showed a minimal effect on the constitutive levels of VEGF mRNA but completely blocked hypoxia-induced VEGF mRNA and protein expression. The inhibitory effects of flavopiridol on VEGF mRNA induction also occurred in the presence of cycloheximide. The transcriptional activation of either a VEGF promoter-luciferase construct or a hypoxia-inducible factor 1 reporter plasmid was not affected by addition of flavopiridol in transient transfection experiments. In contrast, actinomycin D experiments demonstrated that flavopiridol dramatically decreased VEGF mRNA stability. These data provide the first evidence that flavopiridol can affect gene expression by altering mRNA stability. We propose that flavopiridol may interfere with one or more signaling events, leading to hypoxia-induced, protein kinase-modulated, RNA protein binding activity. An important clinical implication of our results is that flavopiridol, presently under investigation in clinical trials, might have antiangiogenic as well as direct antiproliferative effects.

Regulation of BRCA1 by protein degradation.

BRCA1, a tumor suppressor protein implicated in hereditary forms of breast and ovarian cancer, is transcriptionally regulated in a proliferation-dependent manner. In this study, we demonstrate a substantial role for proteolysis in regulating the BRCA1 steady-state protein level in several cell lines. N-acetyl-leu-leu-norleucinal (ALLN), an inhibitor of the proteasome, calpain, and cathepsins, caused BRCA1 protein to accumulate in the nucleus of several human breast, prostate, and melanoma cell lines which express low or undetectable basal levels of BRCA1 protein, but not in cells with high basal expression of BRCA1. Protease inhibition did not increase BRCA1 synthesis, nor change its mRNA level, but it dramatically prolonged the protein's half-life. In contrast to ALLN, lactacystin and PS341, two specific proteasome inhibitors, as well as calpastatin peptide and PD150606, two selective calpain inhibitors, had no effect on BRCA1 stability, whereas ALLM, an effective calpain and cathepsin inhibitor but weak proteasome inhibitor, did stimulate accumulation of BRCA1. Moreover, three inhibitors of acidic cysteine proteases, chloroquine, ammonium chloride and bafilomycin, were as effective as ALLN. These results demonstrate that degradation by a cathepsin-like protease in fine balance with BRCA1 transcription is responsible for maintaining the low steady-state level of BRCA1 protein seen in many cancer cells.

A dominant negative RAS-specific guanine nucleotide exchange factor reverses neoplastic phenotype in K-ras transformed mouse fibroblasts.

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation state depends on the competing action of GTPase Activating Proteins (GAP) and Guanine nucleotide Exchange Factors (GEF). A tryptophan residue (Trp1056 in CDC25Mm-GEF), conserved in all ras-specific GEFs identified so far has been previously shown to be essential for GEF activity. Its substitution with glutamic acid results in a catalytically inactive mutant, which is able to efficiently displace wild-type GEF from p21ras and to originate a stable ras/GEF binary complex due to the reduced affinity of the nucleotide-free ras/GEF complex for the incoming nucleotide. We show here that this 'ras-sequestering property' can be utilized to attenuate ras signal transduction pathways in mouse fibroblasts transformed by oncogenic ras. In fact overexpression of the dominant negative GEFW1056E in stable transfected cells strongly reduces intracellular ras-GTP levels in k-ras transformed fibroblasts. Accordingly, the transfected fibroblasts revert to wild-type phenotype on the basis of morphology, cell cycle and anchorage independent growth. The reversion of the transformed phenotype is accompanied by DNA endoreduplication. The possible use of dominant negative ras-specific GEFs as a tool to down-regulate tumor growth is discussed.

Shear stress downregulation of platelet-derived growth factor receptor-beta and matrix metalloprotease-2 is associated with inhibition of smooth muscle cell invasion and migration.

BACKGROUND: After endovascular injury, smooth muscle cells (SMCs) may be exposed to hemodynamic shear stress (SS), and these forces modulate neointima accumulation. The effect of SS on SMC migration and invasion is unknown, and it was examined in the present study. METHODS AND RESULTS: Bovine aortic SMCs were exposed to laminar SS of 12 dyne/cm(2) for 3 (SS3) or 15 (SS15) hours; control (C3 and C15) SMCs were kept under static conditions. Platelet-derived growth factor (PDGF)-BB-directed SMC migration and invasion were evaluated by a modified Boyden chamber assay with filters coated with either gelatin or reconstituted basement membrane proteins (Matrigel), respectively. SS15 inhibited both SMC migration and invasion (P<0.0001). There was no significant difference between SS3 and C3 cells. Media conditioned with SS15 cells exhibited a reduction in matrix metalloprotease-2 (MMP-2) by zymography and Western analysis. Northern blot analysis revealed no effect of SS15 on MMP-2 mRNA. In contrast, SS15 decreased MMP-2 activator and membrane-type MMP (MT-MMP or MMP-14) mRNA and protein. Furthermore, SS15 decreased PDGF receptor-beta (PDGF-Rbeta) mRNA and protein (P<0.05), and the SS-dependent decrease in PDGF-BB-directed cell migration was rescued by overexpressing PDGF-Rbeta. CONCLUSIONS: SS inhibits SMC migration and invasion via diminished PDGF-Rbeta expression. This effect of SS is associated with decreased MMP-2 secretion and MT-MMP downregulation.

Characterization of nitric oxide-stimulated ADP-ribosylation of various proteins from the mouse macrophage cell line ANA-1 using sodium nitroprusside and the novel nitric oxide-donating compound diethylamine dinitric oxide.

We examined the ability of nitric oxide (NO) to stimulate the ADP-ribosylation of proteins from the mouse macrophage cell line ANA-1. To demonstrate a specific effect of NO, we used a novel compound named diethylamine dinitric oxide (DEA/NO; 1,1-diethyl-2-hydroxy-2-nitrosohydrazine, sodium salt; [Et2NN(O)NO]Na), which releases NO in aqueous solution at neutral pH. DEA/NO stimulated the ADP-ribosylation of at least three cytosolic proteins (M(r) = 28,000, 33,000 and 39,000) from ANA-1 macrophages. The effect of DEA/NO on the ADP-ribosylation of the predominant target p39 was dose dependent (EC50 = 80 microM). Moreover, the effect of DEA/NO was attributed specifically to released NO rather than diethylamine or nitrite. Sodium nitroprusside (SNP) also stimulated the ADP-ribosylation of cytosolic proteins from ANA-1 mouse macrophages. However, SNP exhibited different time- and dose-dependent effects on the modification of p39. NO synthesized via the activity of interferon-gamma plus lipopolysaccharide-induced NO synthase also enhanced the ADP-ribosylation of p39, confirming that the effects of DEA/NO and SNP could be attributed to NO or reactive nitrogen oxide species. Neither pertussis toxin nor cholera toxin stimulated the ADP-ribosylation of p39; however, cholera toxin stimulated the ADP-ribosylation of proteins with approximate molecular weight of 28,000 and 33,000. These data suggest that the induced expression of NO synthase in tumoricidal macrophages may be associated with autocrine and paracrine effects of NO that include the ADP-ribosylation of various proteins. Moreover, these results indicate that DEA/NO and related compounds may be useful as pharmacologic tools for investigating the effects of NO and reactive nitrogen oxide species on macrophages.

Gene therapy for collateral vessel development.

Transfer of the cDNA coding for angiogenic factors represents a novel and promising approach to induce therapeutic angiogenesis and enhance blood flow to ischemic tissues which cannot be revascularized otherwise. This review will focus on therapeutic angiogenesis based on gene transfer techniques for the treatment of myocardial and limb ischemia. The experimental studies demonstrating the angiogenic effect of recombinant growth factors in animal models and in humans, as well as the most promising methods for gene transfer, will be described. Further, gene transfer studies to induce therapeutic angiogenesis will be reviewed to identify critical questions that still need to be answered before gene therapy with angiogenic factors may be considered for routine clinical application.

Tartronates: a new generation of drugs affecting bone metabolism.

In the search for a new class of bone-sparing agents for treating osteopenic disorders, we hypothesized that tartronic acid derivatives, sharing the chemical characteristics both of bisphosphonates and of Gla residues contained in matrix proteins such as osteocalcin, could positively affect bone metabolism. A series of tartronates was therefore tested for their ability to affect bone metabolism. In vitro resorption tests were performed examining pit formation by freshly isolated rat and rabbit osteoclasts plated onto bone slices and exposed to the drugs for 48 h. Tartronates bearing a linear side-chain (DF 1222 and DF 1363A) were the most effective in inhibiting pit excavation in the pM-nM range. Tartronates did not affect osteoclast viability, number, adhesion, or tartrate resistant acid phosphatase activity. Transient cell retraction was observed in osteoclasts plated onto glass and exposed to DF 1222. The maximal effect was seen in cells treated for 4 h at a concentration of 1 pM. DF 1222 accelerated mineralization in cultures of periosteal cells without affecting other osteoblast-like functions. This product was therefore tested in vivo in ovariectomized mice. Bone mass in femur was evaluated, by ash gravimetry, 21 days after ovariectomy. Unfortunately, DF 1222, the most active of tartronates in vitro, was inactive in this test because of its high hydrophilicity and the subsequent too short residence time. On the contrary, its tetrahydropyranyl ether derivative, DF 1363A, endowed with a significantly higher lipophilicity, showed a dose-dependent bone-sparing effect when administered subcutaneously at 10, 30, and 100 mg/kg/die, thus confirming the activity seen in in vitro tests. Because of their feasible parallel effect on both bone resorption and formation, tartronate derivatives may be tested to candidate this class of products for clinical studies.

Wild-type p53 gene transfer inhibits invasion and reduces matrix metalloproteinase-2 levels in p53-mutated human melanoma cells.

The tumor suppressor gene p53 has inhibitory effects on cell growth and angiogenesis and induces apoptosis when overexpressed in melanoma and in a variety of tumor cells by adenovirus-mediated gene transfer. The invasive ability of tumor cells, facilitating local infiltration and metastasis, is related to matrix metalloproteinase levels. In melanoma, matrix metalloproteinase-2 and matrix metalloproteinase-9 have a prominent role in this process. The aim of this study was to evaluate whether wild-type p53 overexpression, obtained by a recombinant adenovirus vector (AdCMV.p53), affects cell invasiveness through modulation of matrix metalloproteinase-2 and matrix metalloproteinase-9. Two human melanoma cell lines were used in this study: the SK-MEL-110, carrying a mutated p53 gene, and the SK-MEL-147, carrying the wild-type p53 gene. SK-MEL-110 cells infected with AdCMV.p53 exhibited decreased invasion capability from day 1 after infection, compared with cells not infected or infected with the control vector AdCMV.Null. This reduced invasiveness was associated with decreased matrix metalloproteinase-2 levels in conditioned media whereas no changes were detected in matrix metalloproteinase-9 secreted levels. No modulation in matrix metalloproteinase-2 mRNA levels was detectable, however, after wild-type p53 gene transfer. Furthermore, protein expression of secreted tissue inhibitor of metalloproteinase-2 was not altered by AdCMV.p53 treatment. In contrast, in SK-MEL-147 cells, AdCMV.p53 did not affect cell invasiveness and levels of secreted matrix metalloproteinase-2. Gene transfer of wild-type p53 inhibited proliferation of both cell lines, showing that also SK-MEL-147 cells respond to wild-type p53 overexpression. This novel mechanism of action of wild-type p53 gene transfer may contribute to its antitumor effect by downregulating cell invasion and matrix metalloproteinase-2 secreted levels in mutated p53 human melanoma cell lines.

A potential role for imaging technology in anticancer efficacy evaluations.

The introduction of imaging methods suitable for rodents offers opportunities for new anticancer efficacy models. Traditional models do not provide the level of sensitivity afforded by these precise and quantitative techniques. Bioluminescent endpoints, now feasible because of sensitive charge-coupled device cameras, can be non-invasively detected in live animals. Currently, the most common luminescence endpoint is firefly luciferase, which, in the presence of O(2) and ATP, catalyses the cleavage of the substrate luciferin and results in the emission of a photon of light. In vivo implantation of tumour cells transfected with the luciferase gene allows sequential monitoring of tumour growth within the viscera by measuring these photon signals. Furthermore, tumour cell lines containing the luciferase gene transcribed from an inducible promoter offer opportunities to study molecular-target modulation without the need for ex vivo evaluations of serial tumour samples. In conjunction with this, transgenic mice bearing a luciferase reporter mechanism can be used to monitor the tumour microenvironment as well as to signal when transforming events occur. This technology has the potential to reshape the efficacy evaluations and drug-testing algorithms of the future.

Intrasubject between-day variability of PD20 methacholine assessed by the dosimeter inhalation test.

The intrasubject between-day variability of the methacholine inhalation test (MIT) was estimated in a group of 30 patients (15 males, mean age 29 yr), representative of a wide range of degrees of nonspecific bronchial responsiveness (from normal to severely increased). In each patient, an MIT with the dosimeter method was carried out on three separate occasions within one week, keeping as constant as possible both the technical and patient-related factors (FEV1 within +/- 5 percent, no recent airway inflammation) and independently from operator-related factors (three tests, three different operators blinded with regard to previous MIT results). On each occasion, twofold increasing doses of methacholine were given from 6.25 to 3,200 micrograms as cumulative doses, at five-minute intervals by means of a dosimeter Me.far MB3 (nebulization time 0.8 +/- 0.2s, output 5 +/- 0.2 microliters/puff). The FEV1 was measured initially and 1.5 and 3 minutes after each inhalation. The test was continued until either a fall of 20 percent or more in FEV1 was obtained or the last dose was reached. The results were expressed in terms of PD20, ie, the dose of methacholine producing a 20 percent fall in FEV1. Under these carefully controlled conditions, the 95 percent confidence intervals (as based on a single determination) corresponded to +/- 0.22 on a log10 scale or, in a more meaningful way, +/- 1.66 fold-difference in PD20 from one visit to the other.

Effects of angiotensin-converting enzyme inhibition on left ventricular geometric patterns in patients with essential hypertension.

Although angiotensin-converting enzyme inhibitors have been shown to affect left ventricular (LV) remodeling favorably in several conditions, it remains unclear whether they can influence LV geometric pattern in hypertension. To address this issue, 122 patients (71 men and 51 women; mean age = 51 +/- 10 years) with mild to moderate hypertension were studied prospectively. All underwent clinical evaluation and Doppler echocardiography at entry and more than 2 years of quinapril therapy (10-40 mg/day). According to either LV mass (normal if < 131 g/m2 for men or < 100 g/m2 for women) or the ratio of LV posterior wall thickness to diastolic diameter (RWT; normal if < 0.45) at baseline, 58 patients had normal mass and RWT, 18 patients had concentric remodelling (i.e., normal mass but increased RWT), 24 patients had eccentric hypertrophy (i.e., increased mass but normal RWT), and 22 patients had concentric hypertrophy (i.e., increase in both mass and RWT). After 6 months of quinapril therapy, all patients with normal left ventricles showed the maintenance of mass and RWT within normal limits. Patients with concentric remodeling showed no increase in mass but had a significant decrease in RWT. Patients with eccentric hypertrophy exhibited a significant reduction in mass with no substantial change in RWT. Patients with concentric hypertrophy had a significant reduction in both mass and RWT. Changes in LV mass and geometry were maintained during the 2-year period of treatment and were paralleled by improvements in Doppler indices of LV diastolic function in each group. It is concluded that quinapril, with its well-known effects on LV hypertrophy, modifies the LV geometric pattern of hypertensive patients favorably, regardless of the presence of an abnormal LV mass or RWT.

Effectiveness of levodropropizine against cigarette smoke-induced airway hyperreactivity: possible mechanism.

We verified the possible effect of the new antitussive drug levodropropizine on airway hyperreactivity and lung inflammation induced by cigarette smoke exposure in anaesthetized guinea-pigs. Levodropropizine, administered by aerosol at 25 mg/ml for 30 s completely prevented smoke induced airway hyperreactivity. The protective effect was early in onset (3 min) and lasted up to 30 min. The same dose of codeine, administered in the form of an aerosol, decreased the increase in airway responsiveness induced by smoke inhalation slightly but not significantly. In parallel with the functional results, levodropropizine also inhibited the recruitment of inflammatory cells triggered by smoke exposure within the airway lumen. When levodropropizine was administered i.v. to anaesthetized guinea-pigs, it reduced the bronchocontractile effect of capsaicin dose-dependently, whereas it was without effect against substance P-induced bronchoconstriction. These data demonstrate the ability of levodropropizine to counteract the hyperreactive phenomenon and the associated inflammatory event induced by cigarette smoke exposure, an effect which might depend on its capacity to modulate the activation of the peptidergic system.

Gene therapy in peripheral artery disease.

In the last decade, studies of the biological mechanisms underlying angiogenesis, i.e. the development of a new vasculature from pre-existing blood vessels, have suggested a new approach to peripheral obstructive artery disease based on the treatment of ischemic tissues with angiogenic growth factors. As demonstrated by experimental studies in animal models, a therapeutic effect can be reached as the newly formed vascular network, functioning as a biologic by-pass, restores a normal blood supply to the ischemic territories. New techniques of gene therapy proved effective in reaching sustained concentrations of angiogenic factors in the target tissues. This review concerns the pre-clinical background and the results of the early clinical trials of angiogenic gene therapy, which have shown the safety and feasibility of this new approach.

Short-term effects of i.v. injected murine 99MTc-F(ab')2 fragments of an anti-melanoma antibody (HMW-MAA 225.28 S) on haemato-immunological parameters in patients with melanoma.

To evaluate alterations induced by injected murine radiolabelled F(ab')2 fragments of the anti HMW-MAA MoAb 225.28S on the principal haemato-immunological parameters, 32 patients with advanced malignant melanoma were studied. No statistically significant change was found after MoAb administration, but monocytes (3 h after injection) and granular eosinophils (24 h after) were reduced and circulating immune complexes increased (3 h after). No toxic effect or adverse reaction was observed. Therefore, the controlled administration of purified MoAb fragments for diagnostic purposes seems to involve only a very low risk of immediate adverse reactions.