María L. Díez-Marqués

Mice Deficient in Telomerase Activity Develop Hypertension Because of an Excess of Endothelin Production

Background— Telomere shortening has been related to vascular dysfunction and hypertension. In the present study, we analyzed the influence of telomerase deficiency and telomere shortening on arterial pressure (AP). Methods and Results— AP was evaluated in 6-month-old mice lacking the RNA component of the telomerase (terc−/−) at the first generation and third generation (G3). First generation and G3 mice showed higher AP than wild-type (WT) mice. To analyze the mechanisms involved, mean AP and vascular resistance in response to vasoactive substances were measured in G3 and WT mice. These mice showed similar responses to acetylcholine, NG-nitro-l-arginine methyl ester, angiotensin II, and losartan administration. Mean AP did not increase after endothelin-1 (ET-1) administration in G3 mice, but it did in WT animals. Bosentan treatment decreased mean AP only in G3 mice. Serum and urine concentrations of ET-1 were higher in terc−/− than in WT mice. Endothelin-converting enzyme (ECE-1) mRNA expression was higher in terc−/− animals than in the WT group. FR901533, an ECE antagonist, decreased blood pressure in conscious G3 mice. Studies in mouse embryonic fibroblasts from G3 mice suggest that ECE-1 overexpression could be mediated by reactive oxygen species in an AP-1–dependent mechanism, in which some kinases such as PI3-kinase, Akt, erk1/2, and Jun Kinase could be involved. An increased activity of nicotinamide adenine dinucleotide phosphate oxidase seems to be the main source of reactive oxygen species. Conclusions— Mice lacking telomerase activity show hypertension as a result of an increase in plasma ET-1 levels, which is a consequence of ECE-1 overexpression. A direct link between telomerase activity and hypertension is reported.

The leukocyte-endothelial cell interactions are modulated by extracellular matrix proteins.

Background: Endothelium is supported, in normal conditions, by a basement membrane composed, among others, by collagen IV and laminin. Changes in the basement membrane composition could induce changes in endothelial cell modifying their interactions with leukocytes. Methods and Results: Isolated polymorphonuclear cells (PMN) and peripheral blood mononuclear cells (PBMC) were added to cultured human umbilical endothelial cells (HuVEC) previously seeded on collagen IV, collagen I or gelatin. Adhesion of leukocytes to HUVEC and specific cytotoxicity were analysed. PMN adhesion and cytotoxicity were lower whereas those from PBMC were higher when HuVEC were seeded on collagen I, as compared with cells seeded on collagen IV. To analyse the mechanisms involved in these phenomena, P-selectin, ICAM-1, VCAM-1 and MCP- 1 expression were evaluated in HuVEC seeded on the different ECM components. P-selectin and mRNA expression of VCAM-1 were lower in cells seeded on collagen I. By contrast, MCP-1 expression was higher in collagen I. Collagen I-dependent effects were partially prevented when collagen I was treated with pepsin. ILK activity was lower in cells seeded on collagen I, whereas ERK 1/2 activity was enhanced. ILK overexpression reduced ERK 1/2 phosphorylation and this could promote the reduction in P-selectin and the increase in MCP-1. Conclusion: Collagen I decreased ILK activity and this would induce an increase in ERK 1/2 activity in HuVEC. As a consequence, the P-selectin content is diminished and, by contrast, the MCP-1 content is increased. The final effect is a lower recruitment of PMN and a higher adhesion of PBMC.

Arg-Gly-Asp-Ser (RGDS) peptide stimulates transforming growth factor β1 transcription and secretion through integrin activation

Extracellular matrix (ECM) components, through specific peptide motifs such as Arg‐Gly‐Asp (RGD), interact with integrins and can modify the behavior of cells. Transforming growth factor‐β1 (TGF‐β1) is the main cytokine involved in the synthesis of ECM proteins. We analyzed the effect of a RGD‐containing peptide, as Arg‐Gly‐Asp‐Ser (RGDS), on the regulation of TGF‐β1 secretion in cultured human mesangial cells. We found that RGDS increased mRNA expression and secretion of TGF‐β1 by stimulating the TGF‐β1 gene promoter. This effect was dependent on the interaction of RGDS with integrins. We evaluated the signaling pathways implicated in TGF‐β1 production by analyzing the effect of RGDS on kinase‐related integrins. RGDS stimulated tyrosine phosphorylation as well as integrin‐linked kinase (ILK) activity. However, tyrosine kinase inhibitors did not prevent the RGDS effect. In contrast, the inhibition of ILK by cell transfection with a kinase dead‐ILK completely abolished the increased TGF‐β1 secretion and promoter activity in the presence of RGDS. Thus RGDS modulates the secretion of TGF‐β1, probably through increased synthesis by interacting with integrins and activating ILK. This supports a role for ECM components in the regulation of their own secretion.

Telomerase deficiency promotes oxidative stress by reducing catalase activity

Telomere shortening and redox imbalance have been related to the aging process. We used cultured mouse embryonic fibroblasts (MEF) isolated from mice lacking telomerase activity (Terc(-/-)) to analyze the redox balance and the functional consequences promoted by telomerase deficiency. Comparison with wild-type (WT) MEF showed that Terc(-/-) MEF had greater oxidant damage, showing higher superoxide anion and hydrogen peroxide production and lower catalase activity. Restoration of telomerase activity in Terc(-/-) MEF increased catalase expression and activity. TGF-beta1 and collagen type IV levels were higher in Terc(-/-) than in WT MEF. TGF-beta1 promoter activity decreased when Terc(-/-) MEF were incubated with exogenous catalase, suggesting that catalase deficiency is the cause of the TGF-beta1 increase. Similar results were obtained in vivo. Homogenized renal cortex from 6-month-old Terc(-/-) showed higher oxidant capacity, lower catalase activity, greater oxidative damage, and higher TGF-beta1 and fibronectin levels than that from WT mice. In summary, telomerase deficiency reduces catalase activity, determining a redox imbalance that promotes overexpression of TGF-beta1 and extracellular matrix proteins.

Integrin-linked kinase (ILK) modulates wound healing through regulation of hepatocyte growth factor (HGF).

Integrin-linked kinase (ILK) is an intracellular effector of cell-matrix interactions and regulates many cellular processes, including growth, proliferation, survival, differentiation, migration, invasion and angiogenesis. The present work analyzes the role of ILK in wound healing in adult animals using a conditional knock-out of the ILK gene generated with the tamoxifen-inducible Cre-lox system (CRE-LOX mice). Results show that ILK deficiency leads to retarded wound closure in skin. Intracellular mechanisms involved in this process were analyzed in cultured mouse embryonic fibroblast (MEF) isolated from CRE-LOX mice and revealed that wounding promotes rapid activation of phosphatidylinositol 3-kinase (PI3K) and ILK. Knockdown of ILK resulted in a retarded wound closure due to a decrease in cellular proliferation and loss of HGF protein expression during the healing process, in vitro and in vivo. Alterations in cell proliferation and wound closure in ILK-deficient MEF or mice could be rescued by exogenous administration of human HGF. These data demonstrate, for the first time, that the activation of PI3K and ILK after skin wounding are critical for HGF-dependent tissue repair and wound healing.

Losartan-Antioxidant Hybrids: Novel Molecules for the Prevention of Hypertension-Induced Cardiovascular Damage

We report the first examples of a new series of antioxidant-sartan hybrids (AO-sartans), which were made by adding an antioxidant fragment to the hydroxymethyl side chain of losartan. Experiments performed in cultured cells demonstrate that these new hybrids retain the ability to block the angiotensin II effect with increased antioxidant ability. In hypertensive rats, these compounds show properties that suggest they may be more useful than losartan for controlling hypertension and preventing hypertension-induced cardiovascular damage.

Tirofiban increases soluble guanylate cyclase in rat vascular walls: pharmacological and pathophysiological consequences

AIMS Our aim was to evaluate whether tirofiban, which mimics the structure of arginine-glycine-aspartic acid (RGD) peptides, up-regulates soluble guanylate cyclase beta1 subunit (sGC-beta1) expression in vascular smooth muscle cells (VSMCs) and in aorta from rats, and to investigate the pharmacological and pathophysiological consequences of this up-regulation. METHODS AND RESULTS Wistar, Wistar Kyoto, and spontaneously hypertensive rats (SHRs) were used. sGC-beta1 content was assessed by immunoblotting. Arterial pressure was recorded using a tail-cuff sphygmomanometer. Sodium nitroprusside (SNP) and isosorbide dinitrate (IDN) were used as nitric oxide (NO) donors. Tirofiban increased the sGC-beta1 content in VSMCs and in aortic walls from rats after 6 h of treatment. Rats treated with tirofiban experienced a more pronounced decrease in their arterial pressure after acute SNP treatment than vehicle-treated rats. Isolated rat aortic rings incubated with tirofiban showed a higher relaxing response to SNP than control rings as well as an increased sGC-beta1 content and SNP-induced cyclic guanosine monophosphate synthesis. Animals receiving IDN for 1 week showed decreased sGC-beta1 in aortic walls and did not respond to SNP treatment with changes in arterial pressure. Tirofiban restored the decreased sGC-beta1 content in IDN-treated rats and promoted a decreased arterial pressure in response to SNP administration. SHRs showed reduced sGC-beta1 levels, and tirofiban increased these levels and led to a higher response to SNP. CONCLUSION Tirofiban increased the sGC-beta1 content in contractile cells and aortic walls of rats, enhancing the response to SNP and reversing the NO donor tachyphylaxis.

New losartan-hydrocaffeic acid hybrids as antihypertensive-antioxidant dual drugs: Ester, amide and amine linkers

We report new examples of a series of losartan-hydrocaffeic hybrids that bear novel ester, amide and amine linkers. These compounds were made by linking hydrocaffeic acid to the side chain of losartan at the C-5 position of the imidazole ring through different strategies. Experiments performed in cultured cells demonstrate that these new hybrids retain the ability to block the angiotensin II effect and have increased antioxidant ability. Most of them reduced arterial pressure in rats better or as much as losartan.

Discovery of potent calpain inhibitors based on the azolo-imidazolidenone scaffold

A series of new azolopyrimidine-peptide hybrids and indolomethylideneimidazolones were obtained and evaluated as calpain inhibitors. The hybrid compounds were inactive, whereas some members of the initial azolomethylideneimidazolone series showed interesting calpain inhibitory activity. By using 4b as a hit compound, a new series of analogs were synthesized by an efficient synthetic procedure based on a multicomponent reaction followed by an unprecedented reaction at the methylene position of the molecule. The best inhibitor found for calpain I (IC50 = 20 nM) was about 20 times more potent than the hit compound. Studies on 4b showed that its inhibition is consistent with an uncompetitive inhibition mode. This compound did not exhibit cellular toxicity at any of the doses tested (0.1-10 μM) and further studies indicated that it was capable of blockading chemical ischemia induction of apoptosis by preventing sodium azide-dependent calpain activation in intact human kidney tubular epithelial cells. The results of molecular modeling studies rationalized the inhibitory activity found for this series and account, from a structural point of view, for the most active compound identified (4j).