Papers by Francois Alhenc-gelas
Proceedings of the National Academy of Sciences, 2001
Northern Blot, Reverse Transcription (RT)-PCR, and Immunohistochemistry. Total RNA and Northern b... more Northern Blot, Reverse Transcription (RT)-PCR, and Immunohistochemistry. Total RNA and Northern blots were prepared by conventional methods (22) and hybridized with a 0.4-kb tissue kallikrein cDNA probe spanning nucleotides 159-549. RT-PCR (annealing temperature 68°C; 35 cycles) was performed with primers situated in exon 2 (5Ј-GCTTCACCAAATATCAATGT-GGGGGTATC-3Ј) and exon 4 (5Ј-CACACTGGAGCT-CATCTGGGTATTCAT-3Ј) for mKlk1, and with primers located in exon 4 (5Ј-CATATACGAACCCGCAGATGA-TCTCCAGTG-3Ј) and exon 5 (5Ј-CTTTTATCCAAGAGTTA-AACTTAATAAGTTTG-3Ј) for mKlk5. The organs were perfused in vivo with paraformaldehyde before paraffin embedding and immunostaining with an anti-rat tissue kallikrein antibody (23), which was revealed by immunoperoxidase detection Abbreviations footnote: TK, tissue kallikrein; RT, reverse transcription. † To whom reprint requests should be addressed.
Diabetes, 2015
Production of adrenomedullin (ADM), a vasodilator peptide, increases in response to ischemia and ... more Production of adrenomedullin (ADM), a vasodilator peptide, increases in response to ischemia and hypoxia in the vascular wall and the kidney. This may be an adaptive response providing protection against organ damage. We investigated the hypothesis that ADM has a nephroprotective effect in two prospective cohorts of type 2 diabetic patients recruited in France. The highest tertile of plasma MR&proADM (a surrogate for ADM) concentration at baseline was associated with the risk of renal outcomes (doubling of plasma creatinine concentration and/or progression to end&stage renal disease) during follow&up in both cohorts.
Human pathology, 2000
Retained fetal expression of angiotensin I-converting enzyme (ACE, CD143) has recently been shown... more Retained fetal expression of angiotensin I-converting enzyme (ACE, CD143) has recently been shown in intratubular germ cell neoplasms (IGCN) and invasive germ cell tumors (GCT), suggesting the somatic isoform (sACE) as a characteristic component of neoplastic germ cells. We analyzed the distribution of sACE in 159 testicular GCT, including 87 IGCN. sACE protein was determined by immunohistochemistry (MAb CG2) on routinely formalin-fixed and paraffin-embedded tissue sections, supplemented by mRNA expression analysis using in situ hybridization. These data were compared with those obtained by germ cell/placental alkaline phosphatases (PIAP; MAbs PL8-F6 and 8A9) employing an uniform score system for the evaluation of immunoreactivity (IRS; possible values from 0 to 12). Expression of sACE and PIAP was found in all 87 analyzed IGCN (IRS > 4, median IRS of 12). Heterogeneous staining patterns were not related to the type of adjacent GCT but correlated with low expression in adjacent s...
Human pathology, 1988
Angiotensin-I-converting enzyme (ACE) was detected in 18 germinomas, both of testicular or extrat... more Angiotensin-I-converting enzyme (ACE) was detected in 18 germinomas, both of testicular or extratesticular localization, and studied by immunohistochemical methods using specific polyclonal antibodies and by enzyme activity measurements. ACE was also detected in normal human germ cells. On the other hand, it was not present in other types of testicular tumors. Biochemical studies and immunohistochemical findings suggest that at least part of the enzyme is membrane bound. Plasma ACE levels appeared to be normal, indicating that measurement of plasma ACE levels in germinomas would be of little help for the diagnosis and follow-up of patients. However, the apparent specificity of ACE detection in germinomas among germ cell tumors might help in histologic diagnosis, especially for tumors of extragonadal localization.
Archives of Cardiovascular Diseases Supplements, 2014
Elmarakby, Ahmed A., Peter Morsing, and David M. Pollock. Enalapril attenuates endothelin-1-induc... more Elmarakby, Ahmed A., Peter Morsing, and David M. Pollock. Enalapril attenuates endothelin-1-induced hypertension via increased kinin survival. Recent studies have shown that angiotensin-converting enzyme (ACE) inhibitors attenuate endothelin-1 (ET-1)-induced hypertension, but the mechanisms for this effect have not been clarified. Initial experiments were conducted to contrast the effect of the ACE inhibitor enalapril, the combined ACE-neutral endopeptidase inhibitor omapatrilat, and the angiotensin II receptor antagonist candesartan on the hypertensive and renal response to ET-1 in anesthetized Sprague-Dawley rats. Acute intravenous infusion of ET-1 (10 pmol⅐kg Ϫ1 ⅐min Ϫ1 ) for 60 min significantly increased mean arterial pressure (MAP) from 125 Ϯ 8to145Ϯ 8 mmHg (P Ͻ 0.05) and significantly decreased glomerular filtration rate (GFR) from 0.31 Ϯ 0.09 to 0.13 Ϯ 0.05 ml⅐min Ϫ1 ⅐100 g kidney wt Ϫ1 . Pretreatment with enalapril (10 mg/kg iv) before ET-1 infusion inhibited the increase in MAP (121 Ϯ 4 vs. 126 Ϯ 4 mmHg) before and during ET-1 infusion, respectively (P Ͻ 0.05) without blocking the effect of ET-1 on GFR. In contrast, neither omapatrilat (30 mg/kg) nor candesartan (10 mg/kg) had any effect on ET-1-induced increases in MAP or decreases in GFR. To determine whether the effect of enalapril was due to the decrease in angiotensin II or increase in kinin formation, rats were given (1 mg/kg iv), a selective B2 receptor antagonist, with or without enalapril before ET-1 infusion. REF-000359 completely blocked the effect of enalapril on ET-1 infusion (MAP was 117 Ϯ 5 vs. 135 Ϯ 5 mmHg before and during ET-1 infusion, respectively, P Ͻ 0.05). REF-000359 alone had no effect on the response to ET-1 infusion (MAP was 117 Ϯ 4 vs. 144 Ϯ 4 mmHg before and during ET-1 infusion, respectively, P Ͻ 0.05). REF-000359 with or without enalapril had no significant effect on the ability of ET-1 infusion to decrease GFR. These findings support the hypothesis that decreased catabolism of bradykinin and its subsequent vasodilator activity oppose the actions of ET-1 to increase MAP. endothelin; angiotensin-converting enzyme inhibitors; bradykinin receptors; blood pressure; glomerular filtration rate ENDOTHELIN-1 (ET-1) has been described as the most powerful vasoconstrictor yet discovered and produces
Diabetes & Metabolism, 2010
Biological Chemistry, 2004
To investigate the glycosylation of the human bradykinin B2 receptor and the functional significa... more To investigate the glycosylation of the human bradykinin B2 receptor and the functional significance of this modification, we studied receptors mutated at single or multiple combinations of the three potential N-linked glycosylation sites, asparagines N3, N12 and N180, in COS-7, HEK 293 and CHO-K1 cells. Western blot experiments demonstrated that all three extracellular asparagines are glycosylated. The kinetics of bradykinin binding and receptor sequestration remained unchanged after glycosylation had been suppressed. However, the glycosylated receptors were expressed at the cell-surface to a much greater extent than the non-glycosylated receptor and coupling to phospholipase C was less efficient for receptor lacking N-terminal glycosylation. These results indicate that, for the human bradykinin B2 receptor, glycosylation is not required for optimal ligand binding, but plays an important role in cell-surface addressing and receptor function.
Prostaglandins, 1982
Human endothelial cells in culture synthesize prostaglandins and release these products into the ... more Human endothelial cells in culture synthesize prostaglandins and release these products into the culture medium. The major products of arachidonic acid metabolism were identified by high pressure liquid chromatography or thin layer chromatography, and release of prostaglandins was measured by radioimmunoassays. Addition of histamine or bradykinin enhanced release of prostaglandins in both arterial and venous endothelial cells. Other vasoactive compounds including angiotensin II, vasopressin, substance P, epinephrine, norepinephrine, or isoproterenol were ineffective. Release of prostaglandins by histamine was concentration-related, and involved H1 receptors, as determined by addition of histamine antagonists. Incubation of endothelial cells with 14C-arachidonic acid resulted in a time-dependent uptake into cell lipids, where most of the radioactivity was incorporated into phosphatidyl choline and neutral lipids. Endothelial cells released 14C-arachidonic acid as well as 14C-prostaglandins in response to either histamine or bradykinin. The enhanced release of 14C-prostaglandins was inhibited by either indomethacin or mepacrine, but 14C-arachidonic acid release was inhibited only by mepacrine. We conclude that the vasoactive compounds, histamine and bradykinin, stimulate formation of prostaglandins in endothelial cells by the release of arachidonic acid from phospholipids of the cell membrane.
Journal of Pharmacology and Experimental Therapeutics, 2014
Limb ischemia is a major complication of thromboembolic diseases. Diabetes worsens prognosis by i... more Limb ischemia is a major complication of thromboembolic diseases. Diabetes worsens prognosis by impairing neovascularization. Genetic or pharmacological inactivation of the kallikrein-kinin system aggravates limb ischemia in nondiabetic animals, whereas angiotensin I-converting enzyme/kininase II inhibition improves outcome. The role of kinins in limb ischemia in the setting of diabetes is not documented. We assessed whether selective activation of kinin receptors by pharmacological agonists can influence neovascularization in diabetic mice with limb ischemia and have a therapeutic effect. Selective pseudopeptide kinin B1 or B2 receptor agonists resistant to peptidase action were administered by osmotic minipumps at a nonhypotensive dosage for 14 days after unilateral femoral artery ligation in mice previously rendered diabetic by streptozotocin. Comparison was made with ligatured, nonagonist-treated nondiabetic and diabetic mice. Diabetes reduced neovascularization, assessed by microangiography and histologic capillary density analysis, by roughly 40%. B1 receptor agonist or B2 receptor agonist similarly restored neovascularization in diabetic mice. Neovascularization in agonist-treated diabetic mice was indistinguishable from nondiabetic mice. Both treatments restored blood flow in the ischemic hindfoot, measured by laser-Doppler perfusion imaging. Macrophage infiltration increased 3-fold in the ischemic gastrocnemius muscle during B1 receptor agonist or B2 receptor agonist treatment, and vascular endothelial growth factor (VEGF) level increased 2-fold. Both treatments increased, by 50-100%, circulating CD45/CD11b-positive monocytes and CD34(+)/VEGFR2(+) progenitor cells. Thus, selective pharmacological activation of B1 or B2 kinin receptor overcomes the effect of diabetes on postischemic neovascularization and restores tissue perfusion through monocyte/macrophage mobilization. Kinin receptors are potential therapeutic targets in limb ischemia in diabetes.
Archives of Cardiovascular Diseases Supplements, 2014
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2010
Angiotensin I-converting enzyme (ACE; kininase II) levels in humans are genetically determined. A... more Angiotensin I-converting enzyme (ACE; kininase II) levels in humans are genetically determined. ACE levels have been linked to risk of myocardial infarction, but the association has been inconsistent, and the causality underlying it remains undocumented. We tested the hypothesis that genetic variation in ACE levels influences myocardial tolerance to ischemia. We studied ischemia-reperfusion injury in mice bearing 1 (ACE1c), 2 (ACE2c, wild type), or 3 (ACE3c) functional copies of the ACE gene and displaying an ACE level range similar to humans. Infarct size in ACE1c was 29% lower than in ACE2c (P<0.05). Pretreatment with a kinin B2 receptor antagonist suppressed this reduction. In ACE3c, infarct size was the same as in ACE2c. But ischemic preconditioning, which reduced infarct size in ACE2c (-63%, P<0.001) and ACE1c (-52%, P<0.05), was not efficient in ACE3c (-2%, NS, P<0.01 vs. ACE2c). In ACE3c, ischemic preconditioning did not decrease myocardial inflammation or cardiom...
Biochemical pharmacology, Jan 23, 2015
Clinical and experimental studies suggest that pharmacological postconditioning with Cyclosporin ... more Clinical and experimental studies suggest that pharmacological postconditioning with Cyclosporin A (CsA) reduces infarct size in cardiac ischemia and reperfusion. CsA interacts with Cyclophilin D (CypD) preventing opening of the mitochondrial permeability transition pore (mPTP). Tissue Kallikrein (TK) and its products kinins are involved in cardioprotection in ischemia. CypD knockout mice are resistant to the cardioprotective effects of both CsA and kinins suggesting common mechanisms of action. Using TK gene knockout mice, we investigated whether the kallikrein-kinin system is involved in the cardioprotective effect of CsA. Homozygote and heterozygote TK deficient mice (TK(-/-), TK(+/-)) and wild type littermates (TK(+/+)) were subjected to cardiac ischemia-reperfusion with and without CsA postconditioning. CsA reduced infarct size in TK(+/+) mice but had no effect in TK(+/-) and TK(-/-) mice. Cardiac mitochondria isolated from TK(-/-) mice had indistinguishable basal oxidative pho...
Vascular Pharmacology, 2006
Ischemic disease represents the new epidemic worldwide. Animal models of ischemic disease are use... more Ischemic disease represents the new epidemic worldwide. Animal models of ischemic disease are useful because they can help us to understand the underlying pathogenetic mechanisms and develop new therapies. The present review article summarizes the results of a consensus conference on the status and future development of experimentation in the field of cardiovascular medicine using murine models of peripheral and myocardial ischemia. The starting point was to recognize the limits of the approach, which mainly derive from species- and disease-related differences in cardiovascular physiology. For instance, the mouse heart beats at a rate 10 times faster than the human heart. Furthermore, healing processes are more rapid in animals, as they rely on mechanisms that may have lost relevance in man. The main objective of the authors was to propose general guidelines, diagnostic end points and relevance to clinical problems.
Thrombosis and Haemostasis, 2013
Study of mice rendered deficient in tissue kallikrein (TK) by gene inactivation and human subject... more Study of mice rendered deficient in tissue kallikrein (TK) by gene inactivation and human subjects partially deficient in TK activity as consequence of an active site mutation has allowed recognising the physiological role of TK and its peptide products kinins in arterial function and in vasodilatation, in both species. TK appears as the major kinin forming enzyme in arteries, heart and kidney. Non-kinin mediated actions of TK may occur in epithelial cells in the renal tubule. In basal condition, TK deficiency induces mild defective phenotypes in the cardiovascular system and the kidney. However, in pathological situations where TK synthesis is typically increased and kinins are produced, TK deficiency has major, deleterious consequences. This has been well documented experimentally for cardiac ischaemia, diabetes renal disease, peripheral ischaemia and aldosterone-salt induced hypertension. These conditions are all aggravated by TK deficiency. The beneficial effect of ACE/kininase II inhibitors or angiotensin II AT1 receptor antagonists in cardiac ischaemia is abolished in TK-deficient mice, suggesting a prominent role for TK and kinins in the cardioprotective action of these drugs. Based on findings made in TK-deficient mice and additional evidence obtained by pharmacological or genetic inactivation of kinin receptors, development of novel therapeutic approaches relying on kinin receptor agonism may be warranted.
Proceedings of the National Academy of Sciences, 1988
The amino-terminal amino acid sequence and several internal peptide sequences of angiotensin I-co... more The amino-terminal amino acid sequence and several internal peptide sequences of angiotensin I-converting enzyme (ACE; peptidyl-dipeptidase A, kininase II; EC 3.4.15.1) purified from human kidney were used to design oligonucleotide probes. The nucleotide sequence of ACE mRNA was determined by molecular cloning of the DNA complementary to the human vascular endothelial cell ACE mRNA. The complete amino acid sequence deduced from the cDNA contains 1306 residues, beginning with a signal peptide of 29 amino acids. A highly hydrophobic sequence located near the carboxyl-terminal extremity of the molecule most likely constitutes the anchor to the plasma membrane. The sequence of ACE reveals a high degree of internal homology between two large domains, suggesting that the molecule resulted from a gene duplication. Each of these two domains contains short amino acid sequences identical to those located around critical residues of the active site of other metallopeptidases (thermolysin, neutral endopeptidase, and collagenase) and therefore bears a putative active site. Since earlier experiments suggested that a single Zn atom was bound per molecule of ACE, only one of the two domains should be catalytically active. The results of genomic DNA analysis with the cDNA probe are consistent with the presence of a single gene for ACE in the haploid human genome. Whereas the ACE gene is transcribed as a 4.3-kilobase mRNA in vascular endothelial cells, a 3.0-kilobase transcript was detected in the testis, where a shorter form of ACE is synthesized.
Proceedings of the National Academy of Sciences, 2001
Diabetic nephropathy is a major risk factor for end-stage renal disease and cardiovascular diseas... more Diabetic nephropathy is a major risk factor for end-stage renal disease and cardiovascular diseases and has a marked genetic component. A common variant (D allele) of the angiotensin I-converting enzyme (ACE) gene, determining higher enzyme levels, has been associated with diabetic nephropathy. To address causality underlying this association, we induced diabetes in mice having one, two, or three copies of the gene, normal blood pressure, and an enzyme level range (65-162% of wild type) comparable to that seen in humans. Twelve weeks later, the three-copy diabetic mice had increased blood pressures and overt proteinuria. Proteinuria was correlated to plasma ACE level in the three-copy diabetic mice. Thus, a modest genetic increase in ACE levels is sufficient to cause nephropathy in diabetic mice.
Proceedings of the National Academy of Sciences, 1980
Rat kidney contains membrane-bound renin (EC 3.4.99.19) and kallikrein (EC 3.4.21.8). Kallikrein ... more Rat kidney contains membrane-bound renin (EC 3.4.99.19) and kallikrein (EC 3.4.21.8). Kallikrein activity was measured by a spectrophotometric assay and renin by radioimmunoassay. Plasma membrane-bound kallikrein was activated by lysolecithin and by melittin, which was a more potent activator., This activation by mellitin was independent of calcium concentration. Mellitin was, however, a more potent activator of membrane-bound renin in the presence of calcium. Administration of aldosterone to rats for 6 days increased kallikrein activity in the renal homogenate and in the membrane-enriched fractions, whereas renin activity was not affected. It was proposed that kallikrein may also be located on the basal membrane of tubular epithelial cells, where aldosterone can enhance its activity.
Proceedings of the National Academy of Sciences, 2001
Tissue kallikrein is a serine protease thought to be involved in the generation of bioactive pept... more Tissue kallikrein is a serine protease thought to be involved in the generation of bioactive peptide kinins in many organs like the kidneys, colon, salivary glands, pancreas, and blood vessels. Low renal synthesis and urinary excretion of tissue kallikrein have been repeatedly linked to hypertension in animals and humans, but the exact role of the protease in cardiovascular function has not been established largely because of the lack of specific inhibitors. This study demonstrates that mice lacking tissue kallikrein are unable to generate significant levels of kinins in most tissues and develop cardiovascular abnormalities early in adulthood despite normal blood pressure. The heart exhibits septum and posterior wall thinning and a tendency to dilatation resulting in reduced left ventricular mass. Cardiac function estimated in vivo and in vitro is decreased both under basal conditions and in response to adrenergic stimulation. Furthermore, flow-induced vasodilatation is impaired in isolated perfused carotid arteries, which express, like the heart, low levels of the protease. These data show that tissue kallikrein is the main kinin-generating enzyme in vivo and that a functional kallikrein-kinin system is necessary for normal cardiac and arterial function in the mouse. They suggest that the kallikrein-kinin system could be involved in the development or progression of cardiovascular diseases.
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Papers by Francois Alhenc-gelas