Modulation of fibrinolytic and gelatinolytic activity during adipose tissue development in a mouse model of nutritionally induced obesity

A nutritionally induced obesity model was used to investigate the modulation of fibrinolytic and gelatinolytic activity during the development of adipose tissue. Five week old male mice were fed a standard fat diet (SFD, 13% kcal as fat) or a high fat diet (HFD, 42% kcal as fat) for up to 15 weeks. The HFD resulted in body weights of 31 +/- 0.9 g, 38 +/- 2.0 g and 47 +/- 1.9 g at 5, 10 and 15 weeks, respectively; corresponding values for mice on the SFD were 26 +/- 0.6 g, 31 +/- 0.9 g and 31 +/- 1.2 g (all p < 0.001). The weight of the isolated subcutaneous (s.c.) or gonadal (GON) fat after 15 weeks of HFD was 1,870 +/- 180 mg or 1,470 +/- 160 mg, as compared to 250 +/- 58 mg or 350 +/- 71 mg for the SFD (p < 0.001). The HFD induced marked time-dependent hyperglycemia and elevated levels of triglycerides and total cholesterol. The HFD diet also induced a marked hypertrophy of the adipocytes as compared to the SFD, e.g. diameter of 83 +/- 3.0 microns versus 52 +/- 4.2 microns for GON adipocytes at 15 weeks (p < 0.005). Plasma plasminogen activator inhibitor-1 (PAI-1) levels were higher in mice on the HFD as compared to the SFD; they were comparable in extracts of s.c. or GON adipose tissue, whereas at different time points tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activator activity was somewhat lower in the adipose tissues of mice on HFD. Gelatinolytic activity (mainly MMP-2) was detected in s.c. but not in GON adipose tissue of mice on SFD, and decreased on the HFD. In situ zymography on cryosections did not reveal different fibrinolytic activities in s.c. or GON adipose tissues of the HFD as compared to the SFD groups, whereas significantly lower gelatinolytic and higher caseinolytic activities were detected in s.c. and GON tissues of mice on the HFD (p < or = 0.05). The fibrillar collagen content was lower in adipose tissue of mice on HFD. Thus, in this model time-dependent development of adipose tissue appears to be associated with modulation of proteolytic activity.     

Plasminogen binding properties of macrophage inflammatory protein (MIP)-2alpha

The chemokine macrophage inflammatory protein (MIP)-2alpha was identified as a plasminogen binding protein by phage display analysis. MIP-2alpha and a truncated form lacking 5 lysine residues in the COOH-terminal region (mut-MIP-2alpha) were expressed in E. coli and purified to apparent homogeneity. Purified MIP-2alpha but not mut-MIP-2alpha bound specifically to plasminogen, with K(A) of 3.7 X 10(5) M(-1) for the interaction of plasminogen with surface-bound MIP-2alpha. Binding and competition experiments indicated that the interaction involves the region comprising the first 3 kringles of plasminogen and the COOH-terminal lysine-rich domain of MIP-2alpha. Activation of plasminogen bound to surface-associated MIP-2alpha by two-chain urokinase-type plasminogen activator (tcu-PA) was about 2.5-fold more efficient than in solution (catalytic efficiency k(cat)K(M) of 0.1 microM(-1)s(-1), as compared to 0.04 microM(-1)s(-1). In contrast, binding of plasminogen to MIP-2alpha in solution was very weak, as evidenced by the absence of competition of MIP-2alpha with lysine-Sepharose or with human THP-1 cells for binding of plasminogen. In agreement with this finding, addition of excess MIP-2alpha did not affect the main functional properties of plasmin(ogen) in solution, as indicated by unaltered activation rates of plasminogen by tcu-PA or tissue-type plasminogen activator (t-PA), t-PA-mediated fibrinolysis, and inhibition rate of plasmin by alpha2-antiplasmin. Thus, association of MIP-2alpha with surfaces exposes its COOH-terminal plasminogen-binding site, and may result in enhanced local plasmin generation.     

Effect of chronic diuretic treatment on the plasma renin-angiotensin-aldosterone system in essential hypertension.

1 Chronic treatment with a constant dose of hydrochlorothiazide or tienilic acid increases plasma renin activity (PRA) acutely to reach a maximum within the first week. 2 During chronic diuretic therapy from 1 month to 1 year, PRA remained elevated at a rather constant level, though this was somewhat lower than the maximum level reached after 1 week. 3 A significant (P less than 0.01) correlation (r = 0.74) between changes in plasma angiotensin II and renin activity provoked by chronic treatment for 3 months with hydrochlorothiazide and tienilic acid was found. 4 The increase in plasma aldosterone during chronic treatment with hydrochlorothiazide and tienilic acid (1000 mg) is related (r = 0.68; P less than 0.01) to the rise in plasma angiotensin II.     

Deficiency of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) impairs nutritionally induced obesity in mice

Tissue inhibitor of matrix metalloproteinase-1 deficient (TIMP-1(-/-)) mice and wild-type (TIMP-1(+/+)) controls were kept on a standard (SFD) or a high fat diet (HFD) for 15 weeks. At the time of sacrifice, TIMP-1(-/-) mice on HFD had a significantly lower body weight (29 +/- 1.5 versus 41 +/- 1.8 g, p <0.005), and significantly less subcutaneous (0.81 +/- 0.19 versus 1.78 +/- 0.21 g, p <0.05) and gonadal (0.87 +/- 0.17 versus 1.85 +/- 0.18 g, p <0.005) fat mass. These differences were much less pronounced for mice on SFD. On HFD but not on SFD, adipocyte diameters were significantly lower in the adipose tissue of TIMP-1(-/-) mice. Plasma leptin levels in TIMP-1(-/-) mice on HFD were significantly lower as compared to TIMP-1(+/-) mice, and strongly correlated with adipose tissue mass for both genotypes. Staining with an endothelial cell specific lectin revealed a significantly higher blood vessel density, larger stained area and vessel size in adipose tissue of TIMP-1(-/-) mice on HFD. This difference disappeared after normalization to the adipocyte number, suggesting that it does not represent a true enhancement of angiogenesis. Thus, in a murine model of nutritionally induced obesity, TIMP-1 promotes adipose tissue development.     

T-lymphocyte and plasma angiotensin-converting enzyme activity during enalapril and losartan administration in humans

This study evaluated the long-term effects of the angiotensin-converting enzyme inhibitor enalapril and the angiotensin II type 1 receptor antagonist losartan on the angiotensin-converting enzyme activity in T lymphocytes and plasma in patients with essential hypertension. The study was a randomized, placebo-controlled, double-blind, crossover design. Nine patients with sitting blood pressure > or = 95 mm Hg and < or = 105 mm Hg at the end of a 4-week placebo run-in period entered the double-blind phase of the study, which consisted of three 6-week periods during which patients were treated with placebo, enalapril (20 mg, once daily), or losartan (50 mg, once daily) The angiotensin-converting enzyme activity in T lymphocytes was measured as the activity of the degradation of the substrate Hippuryl-His-Leu and as the appearance of the dipeptide His-Leu, which was quantified spectrofluorometrically. Enalapril but not losartan suppressed (p < or = 0.01) the angiotensin-converting enzyme activity in plasma, whereas it stimulated (p < or = 0.05) the angiotensin-converting enzyme activity in circulating T lymphocytes. Our data document induction of angiotensin-converting enzyme in human T lymphocytes during long-term treatment with the angiotensin-converting enzyme inhibitor enalapril. Angiotensin II receptor type 1 antagonism with losartan had no effect on plasma or lymphocytic angiotensin-converting enzyme.     

Enzyme-linked immunosorbent assay for the specific detection of angiostatin-like plasminogen moieties in biological samples

An enzyme-linked immunosorbent assay (ELISA) was developed for the specific detection of human angiostatin-like plasminogen moieties (comprising kringles 1-4) in biological samples. The assay involves prior removal of all other plasminogen moieties by immunoadsorption of diluted samples (to about 10 ng/ml plasminogen) with a mixture of insolubilized MA-42B12 (directed against kringle 5) and MA-31E9 (directed against the proteinase domain). The recovery of angiostatin during this procedure is > or = 95%. Subsequently, angiostatin-like fragments are detected in an ELISA, based on two monoclonal antibodies reacting with nonoverlapping epitopes in the kringle 1-3 domain: MA-36E6 for capture and MA-34D3 for tagging. The assay has a lower detection limit of about 0.1 ng/ml and is performed with intra- and interassay coefficient of variation of 2.4% and 15%. In tumor fluids obtained from cancer patients (n = 10), angiostatin levels ranged between 0.24 and 6.7 microg/ml (1.62+/-0.60 microg/ml; mean+/-S.E.M.) The identity of angiostatin was confirmed by immunoblotting using specific monoclonal antibodies. A weak correlation (r = .66) was observed with the total plasminogen concentration in these samples. This ELISA thus appears suitable for the specific quantitation of angiostatin-like plasminogen moieties in biological samples, and may be useful to study its (patho)physiological relevance.     

Reconstitution of the human 5-HT(1D) receptor-G-protein coupling: evidence for constitutive activity and multiple receptor conformations

The 5-hydroxytryptamine (5-HT) 1D/1B receptors have gained particular interest as potential targets for treatment of migraine and depression. G-protein coupling and other intrinsic properties of the human 5-HT(1D) receptor were studied using a baculovirus-based expression system in Sf9 cells. Coexpression of the human 5-HT(1D) receptor with Galpha(i1), alpha(i2), alpha(i3), or Galpha(o)-proteins and Gbeta(1)gamma(2)-subunits reconstituted a Gpp(NH)p-sensitive, high affinity binding of [(3)H]5-HT to this receptor, whereas the Galpha(q)beta(1)gamma(2) heterotrimer was ineffective in this respect. Competition of [(3)H]5-HT binding by various compounds confirmed that coexpression of the human 5-HT(1D) receptor with Galpha(i/o)beta(1)gamma(2) reconstitutes the receptor in a high affinity agonist binding state, having the same pharmacological profile as the receptor expressed in mammalian cells. Binding of the antagonist ocaperidone to the human 5-HT(1D) receptor in coupled or noncoupled state was analyzed. This compound competed with [(3)H]5-HT binding more potently on the human 5-HT(1D) receptor in the noncoupled state, showing its inverse agonistic character. Ocaperidone acted as a competitive inhibitor of [(3)H]5-HT binding when tested with the coupled receptor form but not so when tested with the noncoupled receptor preparation. Finally, [(35)S]GTPgammaS binding experiments using the inverse agonist ocaperidone revealed a high level of constitutive activity of the human 5-HT(1D) receptor. Taken together, the reconstitution of the human 5-HT(1D) receptor-G-protein coupling using baculovirus-infected Sf9 cells made possible the assessment of coupling specificity and the detection of different binding states of the receptor induced by G-protein coupling or ligand binding.     

Impairment of renal function due to sulphinpyrazone after coronary artery bypass surgery: a prospective double-blind study

In this randomized, double-blind, placebo-controlled trial the renal function was studied in 60 patients recovering from coronary artery bypass surgery treated with a daily dose of 800 mg sulphinpyrazone (SP) or 880 mg acetylsalicylic acid (ASA) or placebo. Serum creatinine level increased (p less than 0.05) during the first 2 days of SP treatment, but returned to its baseline level within 4 days under maintained therapy; during ASA and placebo therapy no significant changes occurred. Serum urea levels decreased (p less than 0.01) during ASA and placebo treatments as time from surgery subsided; the decrease of serum urea level was delayed in the SP group compared with the ASA and placebo groups. Urinary excretion of prostaglandin E2 (PGE2) was significantly decreased (p less than 0.01) during ASA treatment; in the SP group, urinary PGE2 excretion tended also to decrease during the first days of treatment, the decrease being significant only on the 4th day (p less than 0.01). The urinary excretion of kallikrein decreased significantly only in the SP group (p less than 0.01), while the changes in the ASA and placebo group were not significant. We suggest that the rapidly reversible acute renal impairment during SP therapy was probably due to a transient renal ischemia caused by a drug-related decrease in urinary kallikrein excretion rather than by renal prostaglandin inhibition.     

Blood pressure and urinary cations in urban Bantu of Zaire

The 24-hour urinary excretion of sodium, potassium, calcium, and magnesium and their relationship to arterial blood pressure were investigated from December 1983 to May 1984 in a 10% random sample (n = 666) of urban Bantu of Kinshasa, Za?re. In youths aged 10-19 years, blood pressure averaged 109/60 mmHg, and the 24-hour urinary excretion of sodium, potassium, calcium, and magnesium averaged 84 mmol, 30 mmol, 483 mumol, and 2 mmol, respectively. After adjustment for age and body weight, a weak positive association became apparent between diastolic pressure and the urinary sodium to potassium ratio in girls and all youths. In adults aged greater than or equal to 20 years, blood pressure averaged 124/72 mmHg, and the 24-hour urinary excretion of sodium, potassium, calcium, and magnesium averaged 87 mmol, 33 mmol, 828 mumol, and 1.88 mmol, respectively. After adjustment for sex, age, body weight, and pulse rate in all adults, systolic pressure was significantly and positively correlated with urinary sodium excretion and negatively correlated with urinary potassium excretion, while diastolic pressure was weakly associated with urinary calcium excretion. In women, an independent and significant association was also observed between systolic pressure and 24-hour urinary sodium. When instead of the 24-hour urinary excretion of sodium and potassium, the sodium to potassium ratio was considered as an independent variable in multiple regression analysis, both systolic and diastolic pressure were independently and positively related to the sodium to potassium ratio in all adults. These results indicate that in this urban Bantu population, age and body weight are the major predictors of systolic pressure in youths and the major predictors of both systolic and diastolic pressure in adults. The sodium to potassium ratio did contribute to the prediction of blood pressure in girls and when, in youths as well as in adults, both sexes were considered together. Urinary calcium was associated with diastolic pressure only in all adults.