Ricardo de Carvalho Cavalli

Comparative assessment of matrix metalloproteinase (MMP)-2 and MMP-9, and their inhibitors, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in preeclampsia and gestational hypertension

OBJECTIVES To compare the circulating levels of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2, and the MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios in preeclampsia and gestational hypertension with those found in normotensive pregnancies. DESIGN AND METHODS We studied 83 pregnant women (30 healthy pregnant women with uncomplicated pregnancies, 26 with gestational hypertension, and 27 with preeclampsia) and 30 healthy nonpregnant women in a cross-sectional study. MMP and TIMP concentrations were measured in plasma samples by gelatin zymography and ELISA, respectively. RESULTS We found higher plasma pro-MMP-9 levels, and higher pro-MMP-9/TIMP-1 ratios in women with gestational hypertension (95%-CI: 1.031 to 2.357, and 0.012 to 0.031, respectively), but not with preeclampsia, compared with those found in normotensive pregnant women (95%-CI: 0.810 to 1.350, and 0.006 to 0.013, respectively; both P<0.05). We found no significant differences in pro-MMP-2 levels (P>0.05). CONCLUSIONS The higher net MMP-9 (but not MMP-2) activity in gestational hypertension compared with normotensive pregnancy suggests that MMP-9 plays a role in the pathophysiology of gestational hypertension. Conversely, the lack of such alterations in preeclampsia is consistent with the notion that different pathophysiological mechanisms are involved in these hypertensive disorders.

eNOS haplotypes affect the responsiveness to antihypertensive therapy in preeclampsia but not in gestational hypertension

Variations of the endothelial nitric oxide synthase (eNOS) gene have been associated with hypertensive disorders of pregnancy. We examined whether eNOS polymorphisms affect the therapeutic responses of women with gestational hypertension (GH) or preeclampsia (PE). We studied 304 hypertensive pregnant women (152 GH and 152 PE), who were stratified according to clinical and laboratorial parameters of therapeutic responsiveness. We compared the frequencies of three eNOS genetic polymorphisms (T-786C, Glu298Asp and b/a intron 4) in responsive and nonresponsive PE and GH patients. We found no significant differences in genotype or allele distributions when responsive and nonresponsive groups were compared (both PE or GH; all P>0.05). However, the eNOS haplotype distribution differed in PE (but not in GH)-responsive and -nonresponsive groups (P=0.0003). The ‘C-Glu-a’ and ‘T-Asp-a’ hapotypes were associated with responsiveness and nonresponsiveness to therapy, respectively (both P<0.001), thus suggesting that eNOS haplotypes affect the responsiveness to antihypertensive therapy in PE.

Matrix metalloproteinase (MMP)-9 genotypes and haplotypes in preeclampsia and gestational hypertension

BACKGROUND Abnormal production of matrix metalloproteinases (MMPs), especially MMP-9, may play a role in hypertensive disorders of pregnancy. These alterations may result from functional genetic polymorphisms in the promoter region of MMP-9 gene, which are known to change MMP-9 expression. We examined whether 2 MMP-9 polymorphisms (C(-1562)T and (CA)n) and haplotypes are associated with preeclampsia and/or gestational hypertension. METHODS We studied 476 pregnant women: 176 healthy pregnant (HP), 146 pregnant with gestational hypertension (GH), and 154 pregnant with preeclampsia (PE). Genomic DNA was extracted from whole blood and genotypes for C(-1562)T and (CA)n polymorphisms were determined by PCR-RFLP. Haplotype frequencies were inferred using the PHASE ver. 2.1 program. RESULTS For the g.-90(CA)13-25 polymorphism, no significant differences were found in genotype and allele distributions when PE or GH groups were compared with HP group. However, the CT genotype and T allele for g.-1562C>T polymorphism were more commonly found in GH subjects compared with the HP group (both P<0.05). Conversely, we found no differences in genotypes or allele distributions for the g.-1562C>T polymorphism when the PE and the HP groups were compared. No significant differences were found in overall distributions of haplotype frequencies when the GH or the PE group was compared with the HP group. CONCLUSIONS The C(-1562)T polymorphism in MMP-9 gene is associated with gestational hypertension, but not with preeclampsia. These findings may help to explain the higher plasma MMP-9 levels previously reported in GH compared with HP.

Matrix metalloproteinase-9 polymorphisms affect plasma MMP-9 levels and antihypertensive therapy responsiveness in hypertensive disorders of pregnancy.

Abnormal matrix metalloproteinase (MMP)-9 levels may have a role in hypertensive disorders of pregnancy. We examined whether MMP-9 genetic polymorphisms (g.−1562C>T and g.−90(CA)13−25) modify plasma MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 levels and the responses to antihypertensive therapy in 214 patients with preeclampsia (PE), 185 patients with gestational hypertension (GH) and a control group of 214 healthy pregnant (HP). Alleles for the g.−90(CA)13−25 polymorphism were grouped L (low) (<21 CA repeats) or H (high) (⩾21 CA repeats). Plasma MMP-9 and TIMP-1 concentrations were measured by enzyme-linked immunosorbent assay. Plasma MMP-9 concentrations were not affected by genotypes or haplotypes in HP and PE groups, except for the g.−90(CA)13−25 polymorphism: GH patients with the LH genotype for this polymorphism have higher MMP-9 levels than those with other genotypes. The T allele for the g.−1562C>T polymorphism and the H4 haplotype (combining T and H alleles) are associated with GH and lack of responsiveness to antihypertensive therapy in GH. The H2 haplotype (combining C and H alleles) was associated with lack of responsiveness to antihypertensive therapy in PE, but not in GH. In conclusion, our results show that MMP-9 genetic variants are associated with GH and suggest that MMP-9 haplotypes affect the responsiveness to antihypertensive therapy in hypertensive disorders of pregnancy.

Association between matrix metalloproteinase (MMP)-2 polymorphisms and MMP-2 levels in hypertensive disorders of pregnancy

We examined whether two functional polymorphisms (g.-1306C>T and g.-735C>T) in matrix metalloproteinase (MMP)-2 gene are associated with preeclampsia (PE) or gestational hypertension (GH), and whether they modify MMP-2 or tissue inhibitor of metalloproteinase (TIMP)-2 plasma concentrations in these hypertensive disorders of pregnancy. We studied 130 healthy pregnant (HP), 130 pregnant with GH, and 133 pregnant with PE. Genomic DNA was extracted from whole blood and genotypes for g.-1306C>T and g.-735C>T polymorphisms were determined by Real Time-PCR, using Taqman allele discrimination assays. Haplotypes were inferred using the PHASE program. Plasma MMP-2 and TIMP-2 concentrations were measured by ELISA. The main findings were that pregnant with PE have higher plasma MMP-2 and TIMP-2 concentrations than HP (P<0.05), although the MMP-2/TIMP-2 ratios were similar (P>0.05). Moreover, pregnant with GH have elevated plasma MMP-2 levels and MMP-2/TIMP-2 ratios compared to HP (P<0.05). While MMP-2 genotypes and haplotypes are not linked with hypertensive disorders of pregnancy, MMP-2 genotypes and haplotypes are associated with significant alterations in plasma MMP-2 and TIMP-2 concentrations in preeclampsia (P<0.05). Our findings may help to understand the relevance of MMP-2 and its genetic polymorphisms to the pathophysiology of hypertensive disorders of pregnancy. It is possible that patients with PE and the MMP-2 haplotype combining the C and T alleles for the g.-1306C>T and g.-735C>T polymorphisms may benefit from the use of MMPs inhibitors such as doxycycline. However, this possibility remains to be determined.

Infecção urinária na gravidez

Several factors cause urinary tract infection (UTI) to be a relevant complication of the gestational period, aggravating both the maternal and perinatal prognosis. For many years, pregnancy has been considered to be a factor predisposing to all forms of UTI. Today, it is known that pregnancy, as an isolated event, is not responsible for a higher incidence of UTI, but that the anatomical and physiological changes imposed on the urinary tract by pregnancy predispose women with asymptomatic bacteriuria (AB) to become pregnant women with symptomatic UTI. AB affects 2 to 10% of all pregnant women and approximately 30% of these will develop pyelonephritis if not properly treated. However, a difficult-to-understand resistance against the identification of AB during this period is observed among prenatalists. The diagnosis of UTI is microbiological and it is based on two urine cultures presenting more than 10(5) colonies/mL urine of the same germ. Treatment is facilitated by the fact that it is based on an antibiogram, with no scientific foundation for the notion that a pre-established therapeutic scheme is an adequate measure. For the treatment of pyelonephritis, it is not possible to wait for the result of culture and previous knowledge of the resistance profile of the antibacterial agents available for the treatment of pregnant women would be the best measure. Another important variable is the use of an intravenous bactericidal antibiotic during the acute phase, with the possibility of oral administration at home after clinical improvement of the patient. At our hospital, the drug that best satisfies all of these requirements is cefuroxime, administered for 10-14 days. Third-generation cephalosporins do not exist in the oral form, all of them involving the inconvenience of parenteral administration. In view of their side effects, aminoglycosides are considered to be inadequate for administration to pregnant women. The inconsistent insinuation of contraindication of monofluorinated quinolones, if there is an indication, norfloxacin is believed to be a good alternative to cefuroxime. In cases in which UTI prophylaxis is indicated, chemotherapeutic agents are preferred, among them nitrofurantoin, with care taken to avoid its use at the end of pregnancy due to the risk of kernicterus for the neonate.

Increased circulating cell-free hemoglobin levels reduce nitric oxide bioavailability in preeclampsia

Contrasting with increased nitric oxide (NO) formation during healthy pregnancy, reduced NO bioavailability plays a role in preeclampsia. However, no study has examined whether increased NO consumption by enhanced circulating levels of cell-free hemoglobin plays a role in preeclampsia. We studied 82 pregnant women (38 healthy pregnant and 44 with preeclampsia). To assess NO bioavailability, we measured plasma and whole blood nitrite concentrations using an ozone-based chemiluminescence assay. Plasma ceruloplasmin concentrations and plasma NO consumption (pNOc) were assessed and plasma hemoglobin (pHb) concentrations were measured with a commercial immunoassay. We found lower whole blood and plasma nitrite concentrations in preeclamptic patients (-48 and -39%, respectively; both P<0.05) compared with healthy pregnant women. Plasma samples from preeclamptic women consumed 63% more NO (P=0.003) and had 53% higher pHb and 10% higher ceruloplasmin levels than those found in healthy pregnant women (P<0.01). We found significant positive correlations between pHb and pNOc (r=0.61; P<0.0001), negative correlations between pNOc and whole blood or plasma nitrite concentrations (P=0.02; r=-0.32 and P=0.01; r=-0.34, respectively), and negative correlations between pHb and whole blood or plasma nitrite concentrations (P=0.03; r=-0.36 and P=0.01; r=-0.38, respectively). These findings suggest that increased pHb levels lead to increased NO consumption and lower NO bioavailability in preeclamptic compared with healthy pregnant women.

Positive correlations between serum and plasma matrix metalloproteinase (MMP)-2 or MMP-9 levels in disease conditions.

Raquel F. Gerlach, Cesar A. Meschiari, Andrea M. Marcaccini, Ana C.T. Palei, Valeria C. Sandrim, Ricardo C. Cavalli and Jose E. Tanus-Santos* 1 Department of Morphology, Estomatology and Physiology, Dental School of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil 2 Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas, Campinas, Brazil 3 Department of Pharmacology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil 4 Department of Gynecology and Obstetrics, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Brazil

Epistasis among eNOS, MMP-9 and VEGF maternal genotypes in hypertensive disorders of pregnancy

Polymorphisms of the endothelial nitric oxide synthase (eNOS), matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) genes were shown to be associated with hypertensive disorders of pregnancy. However, epistasis is suggested to be an important component of the genetic susceptibility to preeclampsia (PE). The aim of this study was to characterize the interactions among these genes in PE and gestational hypertension (GH). Seven clinically relevant polymorphisms of eNOS (T-786C, rs2070744, a variable number of tandem repeats in intron 4 and Glu298Asp, rs1799983), MMP-9 (C-1562T, rs3918242 and -90(CA)13–25, rs2234681) and VEGF (C-2578A, rs699947 and G-634C, rs2010963) were genotyped by TaqMan allelic discrimination assays or PCR and fragment separation by electrophoresis in 122 patients with PE, 107 patients with GH and a control group of 102 normotensive pregnant (NP) women. A robust multifactor dimensionality reduction analysis was used to characterize gene–gene interactions. Although no significant genotype combinations were observed for the comparison between the GH and NP groups (P>0.05), the combination of MMP-9-1562CC with VEGF-634GG was more frequent in NP women than in women with PE (P<0.05). Moreover, the combination of MMP-9-1562CC with VEGF-634CC or MMP-9-1562CT with VEGF-634CC or-634GG was more frequent in women with PE than in NP women (P<0.05). These results are obscured when single polymorphisms in these genes are considered and suggest that specific genotype combinations of MMP-9 and VEGF contribute to PE susceptibility.

Interethnic differences in ADMA concentrations and negative association with nitric oxide formation in preeclampsia.

BACKGROUND Recent studies have suggested that impaired nitric oxide (NO) formation in preeclampsia may result from increased concentrations of an endogenous NO synthase inhibitor, the asymmetric dimethylarginine (ADMA). However, no previous study has examined whether a negative association exists between ADMA and nitrite concentrations in preeclampsia. Moreover, no previous study has compared ADMA and nitrite levels in black and white preeclamptic pregnant women. METHODS We measured plasma nitrite concentrations using an ozone-based chemiluminescence assay, and plasma ADMA levels using enzyme immunoassays in 94 pregnant (47 healthy pregnant: 16 blacks and 31 whites; and 47 preeclamptic: 14 blacks and 33 whites). RESULTS We found higher ADMA (2.199 + or -0.016 micromol/l vs. 2.112 + or - 0.012 micromol/l; P<0.0001) and lower plasma nitrite levels (102 + or - 7.1 nmol/l vs. 214.8 + or - 26.1 nmol/l; P<0.0001) in preeclamptic compared with healthy pregnant women. Black pregnant had higher ADMA levels than white pregnant women (P<0.05), both in preeclamptic (2.239 + or - 0.020 micromol/l vs. 2.144 + or - 0.019 micromol/l) and in healthy pregnant (2.172 + or - 0.025 micromol/l vs. 2.077 + or - 0.018 micromol/l). Conversely, we found no significant effects of ethnicity on the plasma nitrite levels, both in healthy pregnant and in preeclamptic women (P>0.05). We found a significant negative correlation (P<0.05) between these markers (r=-0.28; P<0.05). CONCLUSIONS Our findings show higher ADMA and lower nitrite levels in preeclamptic compared with healthy pregnant, and the concentrations of these biomarkers are inversely associated. While ethnicity affected ADMA concentrations, no such effect was found with respect to nitrite levels. These results may have important implications for studies on NO biology and therapeutic approaches of preeclampsia.