Gene expression analysis in patients with traumatic anterior shoulder instability suggests deregulation of collagen genes.

Shoulder dislocation occurs in 1-2% of the population. Capsular deformation is a key factor in shoulder dislocation; however, little is known about capsule biology. We evaluated, for the first time in literature, the expression of COL1A1, COL1A2, COL3A1 and COL5A1 in the antero-inferior, antero-superior and posterior regions of the glenohumeral capsule of 31 patients with anterior shoulder instability and eight controls. The expression of collagen genes was evaluated by quantitative reverse transcription-PCR. The expression of COL1A1, COL3A1 and the ratio of COL1A1/COL1A2 were increased in all three portions of the capsule in patients compared to controls (p < 0.05). COL1A2 expression was upregulated in the antero-superior and posterior sites of the capsule of patients (p < 0.05). The ratio of COL1A2/COL3A1 expression was reduced in capsule antero-inferior and posterior sites of patients compared to controls (p < 0.05). In the capsule antero-inferior site of patients, the ratios of COL1A1/COL5A1, CO1A2/COL5A1 and COL3A1/COL5A1 expression were increased (p < 0.05). In patients, COL1A1/COL5A1 was also increased in the posterior site (p < 0.05). We found deregulated expression of collagen genes across the capsule of shoulder instability patients. These molecular alterations may lead to modifications of collagen fibril structure and impairment of the healing process, possibly with a role in capsular deformation.

PAMAM dendrimer derivatives as a potential drug for antithrombotic therapy.

Platelets are anucleated blood cells that play an important role both in the pathogenesis of atherosclerosis and subsequent thrombosis. Dendrimers have attracted great interest in biomedical applications. However, their interactions with cell compounds and compartments are nonselective, thus causing cytotoxicity and hemotoxicity. We derivatized PAMAM G4 and G5 dendrimers to evaluate their interactions with serum metabolites, their effects on the viability of red blood cells, and their antithrombotic properties. PAMAM G4 and G5 derivatives showed better hemocompatibility than the PAMAM G4 and G5 dendrimers without any derivatization (NH2). PAMAM G4-Arginine-Tos and G4-Lysine-Cbz act as potent inhibitors of platelet aggregation induced by ADP. PAMAM G4-Arginine-Tos also showed inhibition of platelet aggregation induced by collagen, TRAP-6 and arachidonic acid. Moreover, G4-Arginine-Tos present inhibition of platelet secretion and thrombus formation under flow conditions. Based on our study, the PAMAM G4-Arginine-Tos derivative is hemocompatible and produces desirable antiplatelet and antithrombotic effects. Thus, this compound has potential applications as an antithrombotic drug or a drug delivery vehicle.

Novel thienylacylhydrazone derivatives inhibit platelet aggregation through cyclic nucleotides modulation and thromboxane A2 synthesis inhibition.

The aim of this study has been to investigate the antiplatelet activity of a new series of thienylacylhydrazone derivatives analogous to the lead compound LASSBio-294 ((2-thienylidene) 3,4-methylenedioxybenzoylhydrazine). The antiplatelet effect was investigated in rabbit and human platelet rich plasma stimulated by arachidonic acid, collagen, ADP and in washed platelet stimulated by thrombin. The effects on the production of cyclic nucleotides and thromboxane A(2) (TXA(2)) in human platelets were also investigated. Compounds LASSBio-785 (N-Methyl (2-thienylidene) 3,4-methylenedioxybenzoylhydrazine), LASSBio-786 (N-Benzyl (2-thienylidene) 3,4-methylenedioxybenzoylhydrazine), LASSBio-787 ((5-Methyl-2-thienylidene) 3,4-methylenedioxybenzoylhydrazine), LASSBio-788 (N-Allyl (2-thienylidene) 3,4-methylenedioxybenzoylhydrazine) and LASSBio-789 ((5-Bromo-2-thienylidene) 3,4-methylenedioxybezoylhydrazine) inhibited platelet aggregation induced by arachidonic acid, collagen and ADP. LASSBio-785, LASSBio-788 and LASSBio-789 presented the higher potency in platelet aggregation induced by arachidonic acid (IC(50) values of 0.3, 0.2 and 3.1 microM, respectively) and collagen (IC(50) values of 0.9, 1.5 and 3.4 microM, respectively), with a 20 to 70-fold increase in potency compared to LASSBio-294. They inhibited the ATP release reaction by 95%, the whole blood aggregation by 35-45% and the TXB(2) production was totally abolished. In addition, they presented a significant effect on bleeding time. Qualitative studies in thrombin-induced washed platelet aggregation in the presence of sodium nitroprusside (SNP) suggested a phosphodiesterase-2 (PDE2) like effect for LASSBio-785, LASSBio-788 and LASSBio-789. They were able to increase the cGMP levels in non-stimulated platelets, in SNP-stimulated platelets and in the presence of 1-H- [1, 2, 4] oxadiazolo [4, 3- a] quinoxalin- 1- one (ODQ). The antiplatelet aggregation activity exerted by thienylacylhydrazone derivatives seems to be related to cyclic nucleotides regulation and TXA(2) synthesis inhibition. The structural modification of compound LASSBio-294 led to the optimization of its pharmacological properties and to the discovery of new potent antiplatelet prototypes with an antithrombotic potential.

Spironolactone prevents cardiac collagen proliferation after myocardial infarction in rats.

1. Aldosterone has been considered a key hormone in the regulation of water, sodium and potassium metabolism, thus influencing blood pressure regulation. More recently, several studies have demonstrated that aldosterone is also produced in extra-adrenal tissues (e.g. the heart), suggesting a paracrine effect for aldosterone, such as to increase collagen synthesis in the heart. 2. Because aldosterone production in the heart increases after myocardial infarction (MI), we investigated the effect of chronic administration of spironolactone, an aldosterone receptor antagonist, in rats after MI compared with the effects produced by losartan and hydralazine. 3. Myocardial infarction was produced in male Wistar rats by surgical ligature of the left coronary artery. Sham-operated animals were used as controls. 4. Spironolactone (20 mg/kg per day), losartan (15 mg/kg per day) or hydralazine (20 microg/kg per day) were administered after MI and used for 1 month. 5. At the end of the treatment period, animals underwent haemodynamic recording (arterial and intraventricular pressures). The collagen content of the heart was evaluated by measuring the hydroxyproline (OH-Pro) concentration in right (RV) and left ventricle (LV) muscle fragments. 6. Infarct size was unaffected by drug treatments. The increased LV end-diastolic pressure observed in MI rats was prevented by losartan and remained unchanged following administration of spironolactone or hydralazine. 7. Losartan prevented RV and LV hypertrophy, as well as collagen proliferation in both ventricles, after MI. The postinfarction hypertrophy observed in RV and LV after MI remained unchanged in infarcted groups treated with spironolactone or hydralazine. 8. The OH-Pro concentration was significantly reduced in LV muscle in the MI group treated with spironolactone (682 +/- 40 vs 557 +/- 21 microg/g for MI vs MI + spironolactone, respectively; P < 0.05), an effect not observed in the hydralazine-treated group. 9. These data suggest that spironolactone prevents collagen proliferation in the surviving myocardium by mechanisms independent of the loading conditions of the heart chambers. Control of postinfarction hypertrophy and collagen accumulation produced by losartan seems to depend on the reduction in loading conditions of the heart chambers.

Collagen-PVP, a collagen synthesis modulator, decreases intraperitoneal adhesions.

BACKGROUND: Adhesion formation in the peritoneal cavity is the most common cause of intestinal obstruction and secondary female infertility. A great effort has been dedicated to reduce adhesion formation because of the associated morbidity and its complications. MATERIALS AND METHODS: This study was designed as a before-after comparative trial and included 14 rabbits, with a weight between 300 and 500 g. All rabbits were appendectomized and 1 month later laparotomized to assess adhesion formation. Rabbits were randomized into two groups, Group I (control group), with no intervention, and Group II (experimental group), treated with an intraperitoneal sponge of collagen-polyvinylpyrrolidone (Clg-PVP). The laparotomy procedure was repeated 1 month later for a new assessment of adhesion formation and histological evaluation by H-E and Masson staining. RESULTS: Histological findings showed abundant infiltrate in the control group, which was mild in the experimental group. With the Masson stain the control group showed a significantly higher amount of collagen than the experimental group and the fibrous tissue was more compact. We found a mean number of adhesions of 3.29 +/- 1.98 for the control group, which decreased to 2.57 +/- 0.79 after the second laparotomy. For the experimental group the mean number of adhesions decreased from 1.86 +/- 0.90 to 0.71 +/- 0.49 after the second laparotomy, with no statistical difference between both groups before Clg-PVP application, but a significant statistical difference after the implantation of Clg-PVP (Student's t test; P < 0.001, two-tailed). CONCLUSION: Collagen-polyvinylpyrrolidone decreases the incidence and size of intraabdominal adhesions after secondary adhesion formation after appendectomy.

Endothelin A receptor blockade decreases expression of growth factors and collagen and improves matrix metalloproteinase-2 activity in kidneys from stroke-prone spontaneously hypertensive rats.

This study hypothesizes that endothelin-1 induces renal damage by increasing expression of growth/inflammatory factors, important in renal fibrosis. Male stroke-prone spontaneously hypertensive rats (SHRSPs) (8-weeks, n = 24) were randomized into three groups: control group, high-salt group (4% NaCl), and salt plus an endothelin A receptor antagonist, BMS 182874 (40 mg/kg/d). After 20 weeks treatment, rats were killed. Messenger RNA (mRNA) expression of renal preproendothelin-1, endothelin A and B receptors, and procollagen I and III was evaluated by reverse transcription polymerase chain reaction. Expression of transforming growth factor (TGF)-beta1 and basic fibroblast growth factor (bFGF) was determined by immunoblotting. Matrix metalloproteinase-2 (MMP-2) activity was measured by zymography. In salt-loaded SHRSPs, preproendothelin-1 mRNA expression was increased 1.6-fold, and endothelin A receptor mRNA expression was decreased (70% of control). Salt-loaded SHRSPs had increased renal expression of TGF-b1 and procollagens. MMP-2 activity was augmented fivefold. BMS decreased (p < 0.01) expression of TGF-beta1, bFGF, and procollagen I and reduced MMP-2 activity. Thus severe hypertension and renal dysfunction in salt-loaded SHRSPs are associated with increased expression of renal endothelin-1, growth factors, and collagen. BMS treatment alleviated these effects, suggesting that nephroprotection by endothelin A receptor blockade is mediated by normalizing expression of growth factors, reducing extracellular matrix deposition, and decreasing MMP activity.

Effect of chronic angiotensin II inhibition on the cardiovascular system of the normal rat.

Previous studies have demonstrated in normal rats that chronic treatment, from weaning to 30 days, with either enalapril or losartan, induced significant changes in cardiovascular structure and function. The present study was performed to assess the effect of either enalapril or losartan on the structure and function of the heart and arteries given to normal rats from weaning until 6 months of age. Animals (n = 48) were divided into three groups: control, enalapril treated, and losartan treated; treated rats received 10 mg/kg/day of drug. Blood pressure, body weight, and water intake were recorded for that time period. DNA, cGMP, collagen, degree of fibrosis, and nitric oxide synthase-NADPH-diaphorase-dependent activity in the heart and arteries were determined. Only significant differences (P < .05) are reported. Blood pressure increased only in control rats (13 +/- 1 mm Hg), enalapril treatment enhanced water intake and reduced the rate of body growth (control, 672.9 +/- 15.4 g; losartan, 692.4 +/- 21.8 g; enalapril, 541.8 +/- 13.8 g). In the heart, DNA (control, 120 +/- 5; losartan, 99 +/- 4; enalapril, 93 +/- 6 microg/100 mg), collagen (control, 2.5 +/- 0.2; enalapril, 1.85 +/- 0.08 microg/100 mg), and fibrosis (control, 3.5 +/- 0.4%; losartan, 2.2 +/- 0.3%; enalapril, 2.1 +/- 0.4%) were reduced by treatment. In the aorta, cGMP (control, 0.15 +/- 0.01; losartan, 0.24 +/- 0.02 pmol/mg), and NADPH-diaphorase (control, 0.114 +/- 0.003; losartan, 0.148 +/- 0.006; enalapril, 0.169 +/- 0.003 as optical density) were enhanced. The enzyme was also higher in the aortic endothelium of treated animals (control, 0.193 +/- 0.010; losartan, 0.228 +/- 0.009; enalapril, 0.278 +/- 0.005). The lower rate of body weight increase, the enhanced water intake, and the reduced cardiac and left ventricular weight attributable to enalapril treatment do not seem to be related to inhibition of the renin-angiotensin system. On the other hand, renin-angiotensin system inhibition induces a protective effect on the heart and aorta through structural and functional changes. Most of this action seems to be exerted through angiotensin II type 1 receptors.

A novel phospholipase A2, BJ-PLA2, from the venom of the snake Bothrops jararaca: purification, primary structure analysis, and its characterization as a platelet-aggregation-inhibiting factor.

This paper describes the isolation and primary structure analysis of a new phospholipase A2 with platelet-aggregation-inhibiting activity from the venom of Bothrops jararaca. The protein, named BJ-PLA2, was isolated by means of ammonium sulfate precipitation and anion-exchange and reversed-phase chromatographies and behaved as a homogeneous single-chain protein on SDS-PAGE. Its amino acid sequence was determined by N-terminal sequencing and analysis of overlapped chemical and proteolytic fragments by automated Edman degradation and mass spectometry determination. BJ-PLA2 consists of 124 amino acid residues and has the structural features of snake venom class II phospholipases A2. Chemical modification with p-bromophenacylbromide caused complete loss of enzymatic activity and partially affected the platelet-aggregation-inhibiting activity of BJ-PLA2.

Inhibitory effect of melatonin on platelet activation induced by collagen and arachidonic acid.

Melatonin, an indolamine synthesized in the pineal gland, is known to have antiprostanoid activity. The inhibition of platelet aggregation induced by melatonin has been proposed to take place through the cyclooxygenase pathway. In the present study, we found that melatonin has a marked inhibitory effect on collagen, arachidonic acid (AA), adenosine diphosphate (ADP), epinephrine, and A23187-induced aggregation in platelet-rich plasma. On the other hand, using metrizamide-filtered platelets resuspended in Tyrode's buffer, melatonin fails to suppress AA-induced platelet aggregation and 14C-5-HT release. Under the same conditions, melatonin inhibits collagen-induced platelet activation; however, the addition of threshold doses of AA (0.3 mM) abrogates this effect. These studies suggest that melatonin also inhibits platelet function at a stage preceding the cyclooxygenase-dependent pathway.

Autoimmune sensorineural hearing loss: a preliminary experimental study.

The aim of this study was to compare cochlear alterations produced by induction of anti-type II collagen antibodies with alterations produced by passive transfer of anticochlear antibodies. Guinea pigs (GP) were used. The anticochlear antibodies were obtained by injecting GP membranous cochlea plus Freund's adjuvant into rabbits. After partial purification of the immunoglobulins, the antibodies (20 mg) were injected intramuscularly into 10 normal GP. A second group of 10 normal GP received intramuscular injections of purified chicken type II collagen (1 mg) plus Freund's adjuvant. A control group of 10 normal GP was studied under the same conditions without any stimulus. The cochlea function was analysed with brainstem evoked audiometry (BERA). The structural study was carried out by immunofluorescent and hematoxylin preparations. The results showed structural alterations in both experimental groups (loss of nucleus in the spiral ganglion); however, significant changes in the BERA were not found. Only increase of the latency of wave I could be seen. These preliminary results support the hypothesis that antibodies to collagen type II may play an important role in human autoimmune sensorineural hearing loss, but the possible existence of other cochlear antigens is discussed.

Influence of co-dergocrine on platelet aggregation.

In the present investigation, we studied the influence of co-dergocrine mesylate (dihydroergotoxin mesylate) on platelet aggregation induced by adrenaline, adenosine diphosphate (ADP) and collagen in vitro. Platelet-rich plasma was incubated with increasing concentrations of co-dergocrine (1.5, 3.0, 6.0, 12.0, 24.0, and 48.0 micrograms/ml). The lowest concentration of co-dergocrine tested (1.5 micrograms/ml) inhibited adrenaline-induced aggregation by 97%. Higher concentrations of the compound caused similar degrees of inhibition. ADP-induced aggregation was only decreased significantly (20%, P less than 0.001) by the highest concentration of co-dergocrine, whereas collagen-induced aggregation was not affected. Our results demonstrate that co-dergocrine selectively inhibits platelet aggregation triggered by adrenaline in vitro.

Influence of co-dergocrine on platelet aggregation

In the present investigation, we studied the influence of co-dergocrine mesylate (dihydroergotoxin mesylate) on platelet aggregation induced by adrenaline, adenosine diphosphate (ADP) and collagen in vitro. Platelet-rich plasma was incubated with increasing concentrations of co-dergocrine (1.5, 3.0, 6.0, 12.0, and 48.0 microng/ml). The lowest concentration of co-dergocrine tested (1.5 microng/ml) inhibited adrenaline-induced aggregation by 97%. Higher concentrations of the compound caused similar degress of inhibition. ADP-induced aggregation was only decreased significantly (20%, P < 0.001) by the highest concentration of co-dergocrine, whereas collagen-induced aggregation was not affected. Our results demonstrate that co-dergocrine selectively inhibits platelet aggregation triggered by adrenaline in vitro

Effect of ketotifen and disodium cromoglycate on human platelet aggregation.

For many years, the mast cell has been considered the principal cell in bronchial asthma. However, there is some evidence to suggest that platelets might be involved in asthma. One of this evidence is the induction by platelet-activating factor or airway hyperreactivity and its inhibition by disodium cromoglycate and ketotifen. Since platelet aggregation is an index of platelet activation, we investigated the effect of these drugs on platelet aggregation induced by ADP, collagen and arachidonate in human platelet-rich plasma. The results obtained show that both drugs inhibit the effect of ADP and collagen. Ketotifen was also shown to inhibit the aggregation induced by arachidonate. The mechanism of the antiasthmatic action of these drugs is at present not clear. If platelet activation as it has been proposed, is involved in asthma, the antiaggregant effect of ketotifen and cromoglycate might be part of its beneficial effect in the treatment of asthma.

Why single daily dose of aspirin may not prevent platelet aggregation.

The effect of different doses of aspirin on the synergistic activity of sodium arachidonate plus platelet activating factor (paf) ADP or collagen in platelet aggregation was studied in human volunteers. Aggregation studies in platelet rich plasma (PRP) showed that aspirinated platelets, unresponsive to arachidonate, when stirred with threshold concentrations of paf, ADP or collagen, reacted differently according to the dose of aspirin and the time elapsed since ingestion. After a single or daily 50 mg dose for 7-10 days independent of elapsed time until blood withdrawal, a complete synergistic activity was obtained. In PRP samples obtained 24 hours after the last aspirin intake, a complete synergistic aggregation was achieved after a single dose or after 7-10 days of 500 mg aspirin ingestion; synergistic effect did not appear when blood was drawn 2.5 hours after intake. The thromboxane B2 concentrations were very low in all samples after PRP stimulation with sodium arachidonate or paf or both. As rationale is that platelet activation in vivo occurs in response to several stimuli, the therapeutic implications of our results is that aspirin may not prevent the agonist potentiation effect when low dose or daily high dose (500mg) are administrated. This may explain the erratic results of most aspirin trials in which this drug was used to suppress platelet function.