Giuseppe Molinaro

Biocompatibility of thermosensitive chitosan-based hydrogels: an in vivo experimental approach to injectable biomaterials.

Chitosan, an amino-polysaccharide obtained from the alkaline deacetylation of chitin, presents an interest as a drug vehicle. Indeed, chitosan solutions containing glycerol-2-phosphate (beta-GP) undergo sol-gel transition at a temperature close to 37 degrees C, which make them suitable for the parenteral administration of drugs. However, before using these chitosan derivatives for biomedical applications, it is important to evaluate their biocompatibility, and particularly to test their inflammatory effects. When injected in the hindpaw of the rat, we have shown that: (i) four chitosan/beta-GP solutions tested triggered a non-specific response, with solutions prepared with chitosans of higher deacetylation degrees yielding a lesser inflammatory reaction and (ii) systemic pretreatment of animals with icatibant, apafant and diphenhydramine did not significantly diminish this response; dexamethasone practically abolished it for all solutions and ketanserine only slightly decreased it in one preparation at two different times. In conclusion, it appears that a higher degree of deacetylation of the chitin chain is desirable for superior biocompatibility.

Aminopeptidase P in individuals with a history of angio-oedema on ACE inhibitors

Angio-oedema is a rare but potentially life threatening side-effect of angiotensin-converting-enzyme (ACE) inhibitor treatment. Identification of individuals at risk of this adverse effect is not possible. Angio-oedema is associated with raised concentrations of bradykinin, which is mainly inactivated by ACE. We assessed the plasma activity of two other enzymes that catabolise bradykinin (aminopeptidase P and carboxypeptidase N) in 39 hypertensive patients with a history of angio-oedema during ACE inhibitor treatment and in 39 hypertensive patients who had never had ACE inhibitor associated side-effects. Patients with previous angio-oedema had a lower plasma activity of aminopeptidase P than did those who never presented with angio-oedema (p=0 003). Our data suggest that low plasma concentrations of aminopeptidase P could be a predisposing factor for development of angio-oedema in patients treated with ACE inhibitors.

A Variant in XPNPEP2 Is Associated with Angioedema Induced by Angiotensin I–Converting Enzyme Inhibitors

Angiotensin I-converting enzyme inhibitors (ACEi), which are used to treat common cardiovascular diseases, are associated with a potentially life-threatening adverse reaction known as angioedema (AE-ACEi). We have previously documented a significant association between AE-ACEi and low plasma aminopeptidase P (APP) activity. With eight large pedigrees, we hereby demonstrate that this quantitative trait is partially regulated by genetic factors. We tested APP activity using a variance-component QTL analysis of a 10-cM genomewide microsatellite scan enriched with seven markers over two candidate regions. We found significant linkage (LOD = 3.75) to a locus that includes the XPNPEP2 candidate gene encoding membrane-bound APP. Mutation screening of this QTL identified a large coding deletion segregating in one pedigree and an upstream single-nucleotide polymorphism (C-2399A SNP), which segregates in the remaining seven pedigrees. Measured genotype analysis strongly suggests that the linkage signal for APP activity at this locus is accounted for predominantly by the SNP association. In a separate case-control study (20 cases and 60 controls), we found significant association of this SNP to ACEi-induced AE (P=.0364). In conclusion, our findings provide supporting evidence that the C-2399A variant in XPNPEP2 is associated with reduced APP activity and a higher incidence of AE-ACEi.

Kinin receptors: functional aspects

Two types of receptors (B1R, B2R) for kinins are defined in mammalian species. Comparative experiments involving recombinant fusion proteins consisting of rabbit B1R or B2R fused to GFP-related proteins are exploited to study the regulation of the response to kinins at the receptor level. The following points will be briefly reviewed and supported by some novel data. (1) The constitutive B2Rs are internalized upon agonist stimulation, but completely recycled to the cell surface; however, B2R destruction can be achieved following limited proteolysis (extracellular trypsin, neutrophil proteases), a plausible down-regulation mechanism in pathology. (2) The inducible B1Rs, stimulated by des-Arg9-kinins, are not phosphorylated nor internalized upon agonist stimulation, but rather undergo a reversible redistribution to caveolae-related rafts. B2Rs are also subjected to this translocation, but only transiently (before endocytosis). (3) Based on the analysis of rabbit aortic smooth muscle cells, B1R induction by cytokines is dependent on nuclear factor KB in rabbit vascular tissue, but exogenous kinins acting on either receptor type do not induce B1R expression.

Bradykinin metabolism in the postinfarcted rat heart : role of ACE and neutral endopeptidase 24.11

The respective role of angiotensin-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) in the degradation of bradykinin (BK) has been studied in the infarcted and hypertrophied rat heart. Myocardial infarction (MI) was induced in rats by left descendant coronary artery ligature. Animals were killed, and hearts were sampled 1, 4, and 35 days post-MI. BK metabolism was assessed by incubating synthetic BK with heart membranes from sham hearts and infarcted (scar) and noninfarcted regions of infarcted hearts. The half-life (t1/2) of BK showed significant differences among the three types of tissue at 4 days [sham heart (114 +/- 7 s) > noninfarcted region (85 +/- 4 s) > infarcted region (28 +/- 2 s)] and 35 days post-MI [sham heart (143 +/- 6 s) = noninfarcted region (137 +/- 9 s) > infarcted region (55 +/- 4 s)]. No difference was observed at 1 day post-MI. The participation of ACE and NEP in the metabolism of BK was defined by preincubation of the membrane preparations with enalaprilat, an ACE inhibitor, and omapatrilat, a vasopeptidase inhibitor that acts by combined inhibition of NEP and ACE. Enalaprilat significantly prevented the rapid degradation of BK in every tissue type and at every sampling time. Moreover, omapatrilat significantly increased the t1/2 of BK compared with enalaprilat in every tissue type and at every sampling time. These results demonstrate that experimental MI followed by left ventricular dysfunction significantly modifies the metabolism of exogenous BK by heart membranes. ACE and NEP participate in the degradation of BK since both enalaprilat and omapatrilat have potentiating effects on the t1/2 of BK.The respective role of angiotensin-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) in the degradation of bradykinin (BK) has been studied in the infarcted and hypertrophied rat heart. Myocardial infarction (MI) was induced in rats by left descendant coronary artery ligature. Animals were killed, and hearts were sampled 1, 4, and 35 days post-MI. BK metabolism was assessed by incubating synthetic BK with heart membranes from sham hearts and infarcted (scar) and noninfarcted regions of infarcted hearts. The half-life ( t ½) of BK showed significant differences among the three types of tissue at 4 days [sham heart (114 ± 7 s) > noninfarcted region (85 ± 4 s) > infarcted region (28 ± 2 s)] and 35 days post-MI [sham heart (143 ± 6 s) = noninfarcted region (137 ± 9 s) > infarcted region (55 ± 4 s)]. No difference was observed at 1 day post-MI. The participation of ACE and NEP in the metabolism of BK was defined by preincubation of the membrane preparations with enalaprilat, an ACE inhibitor, and omapatrilat, a vasopeptidase inhibitor that acts by combined inhibition of NEP and ACE. Enalaprilat significantly prevented the rapid degradation of BK in every tissue type and at every sampling time. Moreover, omapatrilat significantly increased the t ½ of BK compared with enalaprilat in every tissue type and at every sampling time. These results demonstrate that experimental MI followed by left ventricular dysfunction significantly modifies the metabolism of exogenous BK by heart membranes. ACE and NEP participate in the degradation of BK since both enalaprilat and omapatrilat have potentiating effects on the t ½ of BK.

Hypotensive transfusion reactions can occur with blood products that are leukoreduced before storage.

BACKGROUND:  Leukoreduction before storage, rather than bedside white blood cell filtration, is recommended to prevent hypotensive transfusion reactions.

Vasopeptidase inhibitors: a new class of dual zinc metallopeptidase inhibitors for cardiorenal therapeutics

Vasopeptidase inhibitors are a new class of drugs that simultaneously inhibit angiotensin-I-converting enzyme and neutral endopeptidase 24.11, two metallopeptidases responsible for the breakdown of different vasoactive peptides. At least ten vasopeptidase inhibitors are in various stages of development and results obtained in preclinical and clinical studies indicate a promising future for the treatment of hypertension, heart failure and renal disease. However, like angiotensin-I-converting-enzyme inhibitors, vasopeptidase inhibitors are characterized by acute and chronic side-effects that need to be clarified.

Tissue Kallikrein Actions at the Rabbit Natural or Recombinant Kinin B2 Receptors

Abstract—We have examined whether exogenous human tissue kallikrein exerts pharmacological actions via the bradykinin B2 receptor; specifically, whether the protease can bind to, cleave, internalize, and/or activate a fusion protein composed of the rabbit B2 receptor conjugated to the green fluorescent protein (B2R-GFP). The enzyme partially digested the fusion protein at 1 &mgr;mol/L, but not 100 nmol/L, and promoted B2R-GFP endocytosis in HEK 293 cells (≥50 nmol/L). Trypsin and endoproteinase Lys-C, but not plasma kallikrein, also cleaved B2R-GFP. Phospholipase A2 was activated by 50 nmol/L tissue kallikrein in HEK 293 cells expressing B2R-GFP, and this was mediated by the receptor, as shown by the effect of a B2 receptor antagonist and by the lack of response in untransfected cells. However, 500 nmol/L kallikrein elicited a strong receptor-independent activation of phospholipase A2. Tissue kallikrein competed for [3H]bradykinin binding to B2R-GFP only at 1 &mgr;mol/L. A simulation involving kallikrein treatment of HEK 293 cells, pretreated or not with human plasma, evidenced the formation of immunoreactive bradykinin. The enzyme (50 nmol/L) contracted the rabbit isolated jugular vein via its endogenous B2 receptors, but the effect was tachyphylactic, and there was no cross-desensitization with bradykinin effects. Aprotinin prevented all pharmacological responses to tissue kallikrein, indicating that the enzyme activity is required for its effect. The local generation of kinins is a plausible mechanism for the pharmacological effects of lower concentrations of tissue kallikrein (50 to 100 nmol/L); higher levels (0.5 to 1 &mgr;mol/L) can not only initiate the degradation of rabbit B2 receptors but also exert nonreceptor-mediated effects.

Hypotensive reaction during staphylococcal protein A column therapy in a patient with anomalous degradation of bradykinin and Des‐Arg9‐bradykinin after contact activation

BACKGROUND: Hypotensive reactions have occurred in patients taking angiotensin converting enzyme (ACE) inhibitors after infusion of blood previously in contact with negatively charged surfaces capable of generating kinins, which accumulate when ACE, a kininase, is inhibited. A patient with anomalous bradykinin (BK) metabolism who experienced hypotension during extracorporeal staphylococcal protein A (SPA) therapy while on an ACE inhibitor was studied.

ABSENCE OF EFFECT OF CHRONIC ANGIOTENSIN II TYPE 1 RECEPTOR BLOCKADE ON ENDOGENOUS KININ CONCENTRATIONS IN THE CARRAGEENAN-INDUCED PAW EDEMA MODEL IN THE RAT

Abstract The effects of chronic treatment with losartan, an AT 1 receptor antagonist, on the tissue content of bradykinin (BK) and des-Arg 9 -BK and on their pharmacological effects were examined in the carrageenan-induced paw edema model (0.5% solution, 50 μl/paw) in the rat. These effects were compared with those of angiotensin-converting enzyme inhibitors (ACEi). For this purpose, rats were chronically treated with losartan (3, 10 and 30 mg/kg/day) and enalapril or quinapril (1 mg/kg/day). Endogenous BK and des-Arg 9 -BK tissue contents at the site of local inflammation were measured by highly sensitive and specific enzyme immunoassays. Losartan 3 mg/kg/day for 7, 14 and 28 days had no significant effect on carrageenan-induced paw edema, but both losartan 10 and 30 mg/kg/day for 14 days significantly increased the hindpaw volume by 50% at 3 h and by 59% at 5 h. These effects, similar to those measured for ACEi, were inhibited by icatibant, a B 2 kinin receptor antagonist (32.5 nmol/paw), that reduced carrageenan-induced paw edema to the level seen in vehicle-treated rats. In the same model, and contrary to ACEi, losartan 3, 10 and 30 mg/kg/day for 14 days had no significant effect on endogenous BK and des-Arg 9 -BK levels in the local inflammatory site or on circulating and tissue ACE activities. These results show, at least in that model, that the potentiating effects of losartan on carrageenan-induced paw edema are independent of the concentrations of endogenous kinins.