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In addition to their physiologic effects in inflammation and angiogenesis, chemokines are involved in cancer pathology. The aim of this study was to determine whether the chemokine stromal cell-derived factor 1 (SDF-1) induces the growth, migration, and invasion of human hepatoma cells. We show that SDF-1 G protein-coupled receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), and SDF-1 mRNA are expressed in human hepatoma Huh7 cells, which secrete and bind SDF-1. This binding depends on CXCR4 and glycosaminoglycans. SDF-1 associates with CXCR4, and syndecan-4 (SDC-4), a heparan sulfate proteoglycan at the plasma membrane of Huh7 cells, induces the growth of Huh7 cells by promoting their entry into the cell cycle, and inhibits the tumor necrosis factor-alpha-mediated apoptosis of the cells. SDF-1 also reorganizes Huh7 cytoskeleton and induces tyrosine phosphorylation of focal adhesion kinase. Finally, SDF-1 activates matrix metalloproteinase-9, resulting in increased migration and invasion of Huh7 cells. These biological effects of SDF-1 were strongly inhibited by the CXCR4 antagonist AMD3100, by a glycosaminoglycan, heparin, as well as by beta-D-xyloside treatment of the cells, or by c-jun NH(2)-terminal kinase/stress-activated protein kinase inhibitor. Therefore, the CXCR4, glycosaminoglycans, and the mitogen-activated protein kinase signaling pathways are involved in these events. The fact that reducing SDC-4 expression by RNA interference decreased SDF-1-induced Huh7 hepatoma cell migration and invasion strongly indicates that SDC-4 may be an auxiliary receptor for SDF-1. Finally, the fact that CXCR4 is expressed in hepatocellular carcinoma cells from liver biopsies indicates that the in vitro results reported here could be extended to in vivo conditions.

We have synthesised a novel cholesterol-based cationic lipid to promote DNA transfer in cells. This lipid, dimethyl hydroxyethyl aminopropane carbamoyl cholesterol iodide (DMHAPC-Chol) contains a biodegradable carbamoyl linker and a hydroxyethyl group in the polar amino head moiety and is characterised by NMR. Liposomes prepared from this lipid and dioleoyl phosphatidyl ethanolamine (DOPE) in equimolar proportion showed a weak cytotoxicity as revealed by MTT assays and are efficient to deliver plasmids DNA evaluated by the expression of reporter genes in vitro and in vivo. In this paper, we present an original method to determine the lipid concentration based on the colorimetric detection of the colipid DOPE and the measure of the molar ratio DOPE/cationic lipid in the liposome by FTIR spectroscopy. The liposomes and lipid/DNA complexes structures were characterized by transmission electron microscopy (TEM) and by quasi-elastic light scattering (QLS). TEM indicated that the complexes correspond to aggregates containing globular substructures with liposomes size. The method of immuno-gold labelling was used to detect plasmid in the complex and reveals the presence of DNA inside the aggregates. Transfection results showed efficient DNA transfer depending on the charge ratio and liposomes conditioning. Gel retardation results indicated that at a molar charge ratio between X = 1.5 and X = 2.5 (depending on the liposome conditioning), all DNA was taken by liposomes. We showed that conditioning by freeze-drying (lyophilization) facilitates storage and improves transfection efficiency. When the liposomes were lyophilized prior to DNA addition or when the complexes were subjected to freeze-thawing cycles, the obtained complexes showed a transfection with levels enhanced up to four and five-fold respectively for the lyophilized liposomes and freeze-thawed complexes. NMR was used to characterize the modifications under freezing which showed an effect on 31P spectra., (Copyright 2004 Elsevier B.V.)

Tumor cells are elusive targets for standard anticancer chemotherapy due to their heterogeneity and genetic instability. On the other hand, proliferating host endothelial cells (ECs) are genetically stable and have a low mutational rate. Thus, antiangiogenic therapy directed against tumor's ECs should, in principle, improve the efficacy of antitumor therapy by inducing little or no drug resistance. Here we present a gene-directed enzyme prodrug therapy (GDEPT) strategy for targeting the tumor vasculature, using the Escherichia coli nitroreductase (ntr) gene delivery associated with the treatment with the prodrug CB1954. In a first time we demonstrated the ability of the ntr/CB1954 system to induce an apoptotic-mediated cell death on monolayer cultures of human umbilical vein ECs (HUV-EC-C). Then, when ntr-transfected HUV-EC-C cells (HUV-EC-C/ntr(+)) were associated in a three-dimensional (3-D) multicellular nodule model with untransfected B16-F10 murine melanoma cell line, we observed a CB1954-mediated bystander cell killing effect from endothelial to neighboring melanoma cells. To our knowledge, this is the first report indicating that GDEPT-based antiangiogenic targeting may be an effective approach for cancer treatment relied on the spreading of the bystander effect from endothelial to tumor cells.

In vitro and in vivo transgene expression in B16-F10 melanoma cells has been investigated using an original cationic liposome prepared with triethyl aminopropane carbamoyl cholesterol iodide (TEAPC-Chol) as carrier. TEAPC-Chol/DOPE (dioleoyl phosphatidyl ethanolamine) liposomes are unilamellar, very stable and not toxic in the used concentration range. The yield in complexation with plasmid DNA can reach 100% even in the presence of fetal calf serum. The transfection level has been evaluated by luminometric measurements of luciferase expression. With TEAPC-Chol/DOPE (1:1) liposomes, a relatively high transfection level in B16-F10 cells has been observed comparing to commercial reagents. For in vivo assays, the transfection level in tumors induced in Nude mice has been optimized by studying the effects of charge ratio, of the helper lipid and of the injection volume. Results showed that TEAPC-Chol/DOPE (1:1) liposomes have improved 10-fold transfection level versus direct gene transfer of free DNA.

Sodium phenylacetate (NaPa) and some bisphosphonates demonstrated antiproliferative and proapoptotic properties against cancer. We have previously shown that NaPa inhibited cell proliferation of MCF7-ras tumor breast cells both in vitro and in vivo. On the other hand, bisphosphonate activities have only been demonstrated in vitro. Here we evaluated the antitumor effects of a new bisphosphonate, the phenylacetate-bisphosphonate (PaBp), on human breast cancer MCF7 and MCF7-ras cell lines, both in vitro and in vivo. To our knowledge, this is the first report indicating the use of a bisphosphonate derivative as a powerful cytostatic and cytotoxic agent, with proapoptotic and antiangiogenic properties on human breast cancer cells lines, with no animal toxicity.

This paper reports results concerning the transfection of gliosarcoma cells 9L using an original cholesterol-based cationic liposome as carrier. This cationic liposome was prepared from triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol) and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE). The used concentration of liposome was not cytotoxic as revealed by the MTT test. TEAPC-Chol/DOPE liposomes allowed the plasmids encoding reporter genes to enter the nucleus as observed both by electron microscopy and functionality tests using fluorescence detection of green fluorescent protein (GFP) and luminometric measurements of luciferase activity. By changing the cationic lipid/DNA molar charge ratio, optimal conditions were determined. Further, improvement of the transfection level has been obtained by either precondensing plasmid DNA with poly-L-lysine or by adding polyethylene glycol (PEG) in the transfection medium. The optimal conditions determined are different depending on whether the transfection is made with cells in culture or with tumors induced by subcutaneous (s.c.) injection of cells in Nude mice. For in vivo assays, a simple method to overcome the interference of haemoglobin with the chemiluminescence intensity of luciferase has been used. These results would be useful for gaining knowledge about the potential for the cationic liposome TEAPC-Chol/DOPE to transfect brain tumors efficiently.

We investigated by transmission electron microscopy the cellular route in tumor MCF7 cells of DNA labeled with digoxigenin, carried by cationic liposomes (Lip+) prepared from TMAEC-Chol [3 beta(N-(N',N',N'-trimethylaminoethane)-carbamoyl)cholesterol iodide] and TEAPC-Chol [3 beta(N-(N',N',N'-triethylaminopropane)-carbamoyl)cholesterol iodide], two cholesterol-based cationic lipids containing a quaternary ammonium. In a previous work we showed the pathway of cationic lipid/plasmid complexes from the beginning of endocytosis until their entry into the perinuclear area. Beyond this limit, unlabeled exogenous plasmids cannot be distinguished with nuclear DNA. This work dealt with the cellular fate of cationic liposome-vectorized plasmids labeled with digoxigenin using an immunogold procedure. Early after the beginning of transfection (30 min, 1 hr, 5 hr), gold particles were observed only in the cytoplasm and in endosome-like vesicles, whereas after 24 hr gold particles were densely present in the nucleus. These results demonstrate the nuclear localization of plasmids vectorized by the cationic liposomes used. The results are discussed in comparison with transfection efficiency measurements.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that need to be activated before they can function to initiate primary and secondary immune responses in vivo. DCs are also specialized to maintain peripheral tolerance to self after uptake of apoptotic material, likely corresponding to both apoptotic bodies and whole apoptotic cells. Here, we report that murine bone marrow-derived DCs can be activated in vitro by exogenous signals received from apoptotic leukemia cells expressing on the cell surface a model tumor-associated antigen. Injected in vivo, these exogenously activated DCs can function as adjuvants to protect mice against leukemia by stimulating an antigen-specific cellular-mediated cytotoxic immune response. To our knowledge, this is the first report indicating that DCs loaded with apoptotic leukemia cells protect mice against leukemia development.

We have investigated the delivery and the pathway in tumoral MCF7 cells of DNA carried by liposomes prepared from (trimethyl aminoethane carbamoyl cholesterol iodide (TMAE-Chol), a cholesterol-based cationic lipid with a quaternary ammonium on the polar head. The structure of DNA-liposome complexes depends on the length of DNA and on the lipid-DNA charge ratio X. Spherical beads constitute fine structures of the observed complexes even when they appear as aggregates. For oligonucleotide transfer, dissociation from liposomes after transfection, penetration of the oligonucleotides into nuclei, and a long resident time were observed. For plasmid transfer, a correlation between the variation in the transfection level and the ultrastructure of complexes was demonstrated. The results showed a cellular route of lipid/plasmid complexes from the beginning by endocytosis, entrapped into endosomes, released by the latter until entry in the perinuclear area, and then penetration of plasmids inside the nuclei resulting in the observed expression of the beta-galactosidase gene.

Despite extensive surgery for glioblastoma, residual tumor cells always lead to relapse. Gene therapy based on retrovirus-mediated gene transfer of herpes simplex virus type 1 thymidine kinase (HSV-1 TK), which specifically sensitizes dividing cells to ganciclovir (GCV) toxicity, may help eradicate such cells. During glioblastoma surgery, HSV-1 TK retroviral vector-producing cells (M11) were injected into the surgical cavity margins after tumor debulking. After a 7-day transduction period, GCV was administered for 14 days. Safety was assessed by clinical and laboratory evaluations, and efficacy was assessed by MRI-based relapse-free survival at month 4 and by overall survival. Twelve patients with recurrent glioblastoma were treated without serious adverse events related to M11 cell administration or GCV. Quality of life was not negatively influenced by this treatment. Overall median survival was 206 days, with 25% of the patients surviving longer than 12 months. At 4 months after treatment, 4 of 12 patients had no recurrence; their median overall survival was 528 days, compared with 194 days for patients with recurrence (p=0.03 by the log rank test). One patient is still free of detectable recurrence, steroid free and independent, 2.8 years after treatment. Thus, brain injections of M11 retroviral vector-producing cells for glioblastoma HSV-1 TK gene therapy were well tolerated and associated with significant therapeutic responses. These results warrant further development of this therapeutic strategy in brain tumor, including recurrent glioblastoma.

We performed a dose-escalating phase I/II study of retrovirus-mediated herpes simplex virus type 1 thymidine kinase (HSV-1-TK) suicide gene therapy for metastatic melanoma. HSV-1 TK expression, which specifically sensitizes transduced and bystander cancer cells to ganciclovir (GCV) toxicity, was mediated by one (four patients, first dose step) to three (four patients, second dose step) injections of "M11" retrovirus vector-producing cells in melanoma cutaneous nodules. After a 7-day period allowed for cancer cell transduction, GCV was administered for 14 days. Safety was assessed by clinical and laboratory evaluations, and efficacy was assessed by tumor measurements and histology. M11 doses ranged from 76 to 1247 x 10(6) cells. Treatment-related adverse events were mild and transient, limited to inflammatory skin reactions at injection and fever on repeated injections. Plasma GCV was in the active range (>0.2 microg/ml); transgene was detected by polymerase chain reaction in three of six patients; treated tumor size was moderately affected under GCV as compared with untreated tumors, although 2 weeks after GCV administration important (>50%) treated-tumor necrosis was evidenced on histology in three of eight patients. All patients showed disease progression on long-term follow-up. Thus, M11-mediated HSV-1 TK gene therapy was well tolerated over a wide dose range. The limited tumor response is likely to be related to poor gene transfer efficiency. However, necrosis following GCV administration in transduced tumors indicates a potential for treatment efficacy.

We examined the efficacy of suicide gene therapy for nitrosomethylurea-induced mammary tumors in rats. Individual tumors were directly injected with a retrovirus-producing cell line that releases retroviral vectors that transduce the herpes simplex virus type 1 thymidine kinase (HSV1-TK) gene. HSV1-TK specifically converts the nucleoside analogue ganciclovir (GCV) into a toxic metabolite. Compared to control rats receiving saline, we observed a significant tumor regression of the injected tumors following GCV administration, accompanied by a stromal inflammation and an extensive lymphocyte infiltration invading the tumor epithelium. It is noteworthy that the neighboring uninjected tumors also regressed, demonstrating the occurrence of a distant bystander effect. This is the first demonstration that HSV1-TK/GCV can efficiently treat multiple solid tumors directly generated from an epithelial tissue.

We recently described a novel nonviral/viral vector for gene transfer, the plasmovirus (Noguiez-Hellin P, Robert-le Meur M, Laune S, et al. C R Acad Sci Paris, Sciences de la Vie. 1996;319:45-50; Noguiez-Hellin P, Robert-le Meur M, Salzmann J-L, et al. Proc Natl Acad Sci USA. 1996;93:4175-4180). Plasmoviruses are plasmids capable of expressing all the viral genes required for generating infectious particles and packaging a defective genome containing a transgene. Transfected as plasmids, plasmoviruses transform the transduced cells into packaging cells that release infectious replication-defective retrovirus vectors (RV) containing a transgene, which are capable of infecting nearby cells. We previously showed that such a vector can efficiently "propagate" the transgene after transfection. Here we examine in greater detail the different steps of plasmovirus replication in vitro in human (143 B TK-) and murine (NIH 3T3 TK-) cells. Molecular-biological analysis revealed plasmovirus-coded protein expression starting from 24 hours post-transfection, followed by the detection of infectious RV 48 hours post-transfection. The gag proteins were correctly processed in the released particles. Electron microscopic analysis revealed typical type C particles. Nonintegrated plasmovirus DNA was not toxic for the cells and could be detected for at least 14 days post-transfection. While the transfected gag gene and the transgene could also be detected throughout this period, we observed that env-coded proteins decreased after 72 hours post-transfection. Nevertheless, the production of RV resulted in the propagation of the transgene in the culture, with stable integration of plasmovirus proviral DNA into the host genome of infected cells. We show that this propagation results in a major improvement in therapeutic efficacy using an HSV1-TK transgene and ganciclovir treatment, when compared to that of plasmovirus constructs that cannot propagate. Altogether, these results demonstrate the functionality of this gene transfer method and suggest that improvements in the vector design enhance its efficacy.

We have generated a chimeric gene transfer vector that combines the simplicity of plasmids with the infectivity and long-term expression of retroviruses. We replaced the env gene of a Moloney murine leukemia virus-derived provirus by a foreign gene, generating a plasmid that upon transfer to tumor cells generates noninfectious retroviral particles carrying the transgene. We added to this plasmid an independent expression cassette comprising a cytomegalovirus promoter, an amphotropic retroviral envelope, and a polyadenylylation signal from simian virus 40. These constructs were designed to minimize the risk of recombination generating replication-competent retroviruses. Their only region of homology is a 157-bp sequence with 53% identity. We show that the sole transfection of this plasmid in various cell lines generates infectious but defective retroviral particles capable of efficiently infecting and expressing the transgene. The formation of infectious particles allows the transgene propagation in vitro. Eight days after transfection in vitro, the proportion of cells expressing the transgene is increased by 10-60 times. There was no evidence of replication-competent retrovirus generation in these experiments. The intratumoral injection of this plasmid, but not of the control vector lacking the env gene, led to foci of transgene-expressing cells, suggesting that the transgene had propagated in situ. Altogether, these "plasmoviruses" combine advantages of viral and non-viral vectors. They should be easy to produce in large quantity as clinical grade materials and should allow efficient and safe in situ targeting of tumor cells.

Therapeutic gene transfer has progressed quite rapidly in recent years. Noticeably, it has now reached the clinical stage in both fields of inherited and acquired diseases. Numerous studies of liver-targeted gene therapy and cancer gene therapy have supported the hope that such innovative approaches may be of help in the treatment of primary or secondary liver tumors. In this article, the main strategies of experimental, hepatic cancer gene therapy in the prospect of a clinical use are reviewed.

This work was aimed at generating a novel system for gene transfer to tumor cell, combining the advantages of non-viral gene transfer methods with those of transfer by recombinant retroviruses. We replaced the env gene of an infectious Moloney murine leukemia provirus with the gene coding for the thymidine kinase of Herpes Simplex Virus 1 (HSV1-TK). The sole transfection of this construction allows the production of viral particles, and the encapsidation of a viral genome carrying transgene. We show that this gene is expressed at a level sufficient for conferring sensitivity to ganciclovir, a nucleoside analog that is metabolised in a toxic compound by HSV1-TK. We also show that the complementation of this recombinant defective provirus with a gene coding for a retroviral envelope, either expressed constitutively by the transduced cell, or by co-transfection, leads to the formation of infectious viral particles capable of transducing HSV1-TK into tumor cells.

The herpes simplex virus type 1 thymidine kinase (HSV1-TK) converts nontoxic nucleoside analogs such as ganciclovir into phosphorylated compounds that act as chain terminators and specifically kill dividing cells. This property could be exploited for the treatment of tumors that are made up of rapidly dividing cells invading a nonproliferating tissue. For this purpose, specific expression of the suicide gene into dividing tumor cells can be further targeted by using retroviral-mediated gene transfer. We investigated whether the direct intratumoral transduction of a suicide gene might induce the elimination of malignant solid tumors. Rats with established macroscopic liver metastases were given an intratumoral injection of packaging cells producing either HSV1-TK- or lacZ-expressing recombinant retroviral particles. All rats were next treated with ganciclovir. A dramatic regression of the tumor volume was observed in the HSV1-TK-treated animals. The residual tumors were mostly made up of a massive fibrotic reaction, with the mean cancer cell mass being reduced approximately 60-fold compared to controls. In some animals, the residual tumors were devoid of cancer cells. This treatment efficacy appears in part due to a "bystander effect" in which phosphorylated ganciclovir could be transferred from cell to cell and to an active local immune reaction evidenced by massive infiltration of the tumors by macrophages and both CD4+ and CD8+ lymphocytes. This efficient therapeutic approach might be an ultimate treatment for disseminated liver metastases in humans and could also be applied to treatment of a large variety of solid tumors.

The effects of long-term angiotensin-converting enzyme (ACE) inhibitor treatment with perindopril 2 mg/kg/day, by gavage, for 3 months on the mechanical function and structure of large arteries were studied in adult spontaneously hypertensive rats with established hypertension. Hemodynamic parameters, including instantaneous aortic blood flow and pressure, were recorded under anesthesia at the end of the treatment period. Systemic arterial compliance was calculated from aortic pressure and flow recordings; passive mechanical properties of the in situ localized carotid artery were measured. Histologic and morphologic parameters of the aortic media, including cross-sectional area and thickness, size, and density of smooth muscle nuclei and of elastin and collagen fibers, were measured using an automated image analysis system. ACE inhibitor treatment significantly decreased mean arterial pressure (-27%, p < 0.001) and total peripheral resistance (-30%, p < 0.05) while cardiac output was increased (29%, p < 0.05). Systemic arterial compliance and carotid compliance were both increased by treatment (63%, p < 0.05, and 83%, p < 0.05, respectively). Morphometric assessment of vascular structure showed that ACE inhibitor treatment significantly decreased medial cross-sectional area (-36%, p < 0.001) and thickness (-16%, p < 0.001) by affecting smooth muscle cell hypertrophy (nucleus size decreased by 26%, p < 0.05) without changes in smooth cell number. Collagen density was decreased by treatment (-42%, p < 0.05), whereas elastin density was not affected by treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of this study was to investigate the propensity to develop cardiac arrhythmias during an acute period of ischemia between normal and hypertrophied (by means of a swimming training regimen) rat hearts. We used the coronary artery ligation in vivo technique which induced the occurrence of cardiac arrhythmias in rats that was followed by the determination of the occluded zone size. This study was coupled to an in vitro study using a two-compartment tissue bath in which half of the ventricular preparation was exposed to normal conditions and the other to ischemic conditions (low pH, hypoxia, and hyperkalemia). We also measured the collagen content and the DNA/protein ratio of the hearts. Twenty-eight male Wistar rats submitted to an eight-week swimming training (SWT) and twenty-eight cage-confined matched rats were used for the studies. SWT resulted in a 14% decrease in mean body weight and an 8% increase in absolute heart weight. We also observed a resting bradycardia in the trained animals and blood pressure remained unchanged between the two groups. Collagen content was unchanged and DNA/protein ratio was lower in the left ventricle of trained animals. During a 30-min period of coronary artery ligation, SWT rats demonstrated fewer ischemia-induced arrhythmias as compared to controls. The size of the zone affected by the vasal occlusion was lower in trained animals. Electrophysiological data recorded in the two-compartment bath showed a marked prolongation of action potential duration and refractory period in the SWT rat hearts. During the 15-min period of in vitro ischemia there was a global alteration of all electrophysiological parameters which did not differ between the two groups. Our data support the hypothesis that resting bradycardia and decrease in ischemic zone size may be involved in the arrhythmogenic protection observed in hypertrophied hearts of swimming rats after an acute ligation of the left coronary artery. Our results also indicate that cardiac hypertrophy, as defined by quantitative changes in cardiac mass or by the electrophysiological alterations that are related to its development, is not necessarily associated with an increased risk for the occurrence of arrhythmias.

We investigated whether the transduction of a suicide gene might induce the elimination of malignant solid tumours. BDIX male rats were given an intra-hepatic injection of a mixture containing DHDK12 tumor cells and xenogeneic fibroblasts. The latter were producing either the herpes simplex virus type 1 thymidine kinase HSV1-TK- or nls-lac Z(control)-expressing recombinant retroviral particles. 5 days later, a time at which the tumor is macroscopic, all the rats were treated with ganciclovir, a nucleoside analog that is metabolized by HSV1-TK into a toxic compound. After 5 days of treatment, a dramatic reduction in tumour volume was noted in the HSV1-TK group. These results delineate a new therapeutical strategy for the treatment of disseminated liver metastases or of a large variety of solid malignant tumours.

The role of genetically determined immune attack and blood pressure in graft rejection-induced arterial wall injury and response was assessed by studying the compliance and changes in wall structure of aortic isografts and allografts in normotensive (Wistar-Kyoto [WKY]) and hypertensive (spontaneously hypertensive [SHR]) rats. Six groups of 8-week-old rats were compared: sham-operated in both strains, isografts, and allografts between the two strains (SHR aortas grafted in WKYs, designated SWs; WKY aortas grafted in SHRs, designated WSs; isografts in SHRs, designated SSs; and isografts in WKYs, designated WWs). Each arterial graft was studied 8 weeks after transplantation for volume and compliance (pressures of 75-175 mm Hg) under basal conditions. The amounts of collagen, elastin, and nuclei in the media and intima of the walls of control and grafted aortas were quantified morphometrically. Isografts and controls had the same mechanical characteristics under basal conditions: the arterial volume and arterial compliance of hypertensive rats were lower than those of normotensive rats (p less than 0.001). Allografts had a greater initial volume (p less than 0.001) and a lower compliance (p less than 0.001) than did isografts. Allografts in SHRs (SSs) were initially dilated, whereas allografted WKYs (WWs) were not. There was intimal proliferation in hypertensive isografts (14 +/- 0.77 microns) and in both types of allografts (WS, 69 +/- 1.55 microns; SW, 44 +/- 1.81 microns); nucleus density was higher in hypertensive allografts (WS) than in normotensive allografts (SW); and collagen density was also higher in SW than in WS allografts. Allografts had decreased medial thickness and decreased smooth muscle cell density.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemodynamic and structural changes of the heart and large arterial vessels were studied in normotensive and spontaneously hypertensive rats following 12 weeks administration of converting enzyme inhibitor (perindopril, 2 mg/kg daily by gavage). In both strains, a significant blood pressure reduction was observed. In normotensive rats, the hemodynamic changes involved significant increase in systemic arterial compliance whereas slight changes in left ventricular weight and aortic medial thickness were observed. In hypertensive rats, the increase in compliance was relatively small, whereas there was a major reduction in medial thickness. Furthermore, the reduction of the media thickness was much more pronounced than that of the left ventricular hypertrophy. The present results suggest that the cardiac and arterial changes observed following long term converting enzyme inhibition do not strictly parallel the blood pressure changes in hypertensive rats. Dissociation between cardiac and arterial changes may be observed.

Relative 125I-albumin concentration was measured in vivo in the aortic media of sham-operated (n = 10) and hypertensive (two-kidney, one clip) rats, untreated (n = 8) or treated (n = 10) by an angiotensin converting enzyme inhibitor (CEI, Trandolapril). Blood pressure was acutely lowered to a normal level at the time of the experiment in hypertensive rats (n = 7) to separate the direct effect of increased pressure from the effect of pressure-induced structural changes. Relative tissue concentration profiles of labeled albumin across the media were obtained using a serial frozen-sectioning technique. In hypertensive rats, the mean medial albumin concentration decreased by 35% in the ascending arch and 32% in the descending arch (p less than 0.01). When blood pressure was acutely lowered in hypertensive animals, this value decreased further by 56% in the ascending arch, 48% in the descending arch (p less than 0.01), and 22% in the thoracic aorta (p less than 0.05) as compared with controls. The medial thickness in hypertensive rats was significantly increased (more in the ascending arch than in the rest of the aorta). Four-week CEI treatment reversed hypertension and medial thickening, but the mean medial albumin concentration remained significantly lower in the arch (by 36% in the ascending part and 40% in the descending part, p less than 0.01). The collagen content in the thoracic aorta was significantly increased in hypertensive rats (by 40%, p less than 0.01) and remained increased (by 29%, p less than 0.01) after CEI treatment. These results suggested that the hypertension-induced structural changes might reduce the medial distribution volume for albumin, whereas elevated blood pressure per se tended to enhance albumin concentration within the media. However, the net result of chronic hypertension was a reduction of the mean medial albumin concentration. The aortic arch appeared to be more affected than the rest of the aorta. Fiber content, more than medial thickness, might be responsible for the observed differences in albumin concentration. Lowering of blood pressure seemed to be insufficient to restore normal albumin concentration profiles and perhaps those of other macromolecules. This finding may be relevant in evaluating some of the complications associated with hypertension.

We studied whether electrophysiological response to ischemia could be different in hypertrophied left ventricle of spontaneously hypertensive rats (SHRs) and in normal left ventricle of normotensive Wistar-Kyoto (WKY) rats. For that purpose, we used a two-compartment tissue bath in which one-half of a left ventricular strip was exposed to normal conditions and the other part to ischemic conditions (low pH, hypoxia, and hyperkalemia). Electrical activity was recorded using standard microelectrodes in normal and hypertrophied preparations. Under basal conditions, the action potential duration (APD) and effective refractory period (ERP) were longer in SHRs than in WKY rats (118 +/- 9 ms vs 81 +/- 3 ms, p less than 0.05 and 90 +/- 9 ms vs. 74 +/- 4 ms, p less than 0.05, respectively). During ischemia, the APD and ERP changed in opposite ways in both groups and we observed the development of postrepolarization refractoriness that was greater in hypertrophied than in normal hearts. Maximum upstroke velocity (Vmax) values were 229 +/- 12 and 227 +/- 10 V/s in the SHR and the WKY preparations, respectively. Three minutes after the induction of ischemia, Vmax values decreased to 46 +/- 7 V/s in SHRs and to 106 +/- 12 V/s in WKY rats. A significant increase in subendocardial collagen density was measured in SHR left ventricle compared to WKY rats (4.39 +/- 0.34% vs. 1.66 +/- 0.15%, p less than 0.05), which might partly explain the impaired conduction observed in hypertrophied preparations during ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemodynamic and structural changes of the heart and large arterial vessels were studied in normotensive and spontaneously hypertensive rats following 12-week administration of converting enzyme inhibitor (perindopril, 2 mg/kg daily by gavage). In both strains, a significant blood pressure reduction was observed. In normotensive rats, the hemodynamic changes involved significant increase in systemic arterial compliance, whereas slight changes in left ventricular weight and aortic medial thickness were observed. In hypertensive rats, the increase in compliance was relatively small, whereas there was a major reduction in medial thickness. The reduction of the media thickness was much more pronounced than that of the left ventricular hypertrophy. In both strains, the collagen density in the subendocardial layers of the left ventricle was significantly decreased in treated vs untreated groups. The isomyosine profile of the left ventricular muscle was also modified by ACE inhibition with an increase in the V1 form and a decrease in the V3 form. The present results suggest that the cardiac and arterial changes observed following long-term converting enzyme inhibition do not strictly parallel the blood pressure changes in hypertensive rats. Dissociation between cardiac and arterial changes may be observed.

An experimental in vivo model of aortic aneurysm was established by perfusing an isolated segment of rat abdominal aorta with pancreatic elastase. Ten rats were used in each protocol. Saline-perfused aortas developed no aneurysmal dilations. Elastase-perfused aortas contained aneurysms in the perfused area and a total loss of elastic tissue. Control aortas contained no elastic tissue lesions. There was a quantitative relation between the amount of elastase perfused and aneurysm formation: 1-2 units induced neither macroscopic nor microscopic lesions; 3-6 units induced microscopic elastic tissue damage without macroscopic aneurysm; and more than 6 units produced aneurysmal dilation in all cases. In situ elastase secretion by macrophages was induced by perfusing rat aortas with thioglycollate-activated macrophages or with thioglycollate alone. There was aortitis without true aneurysm and a total loss of elastic tissue in the vicinity of activated macrophages within the aortic media. Perfusion of infra-aneurysmal amount of elastase (1 or 2 units) or thioglycollate plus plasmin (2 units) always induced a large aneurysm, whereas plasmin alone induced neither macroscopic nor microscopic lesions. These morphological results were supported by the significantly elevated elastolytic activity within the aortic wall of animals perfused with thioglycollate plus plasmin 9 days, after perfusion (207.6 +/- 54.8 micrograms elastin-rhodamine lysed/18 hr; control rats, 25.43 +/- 11.13). The results suggest that the presence of elastase within the aortic media leads to aneurysm formation. Activated macrophages within the aortic media may be responsible for elastase secretion and elastic tissue destruction. Plasmin may enhance elastase activity and aggravate the aneurysmal lesion.

The vascular smooth muscle cell of the arterial media plays a predominant role in functional and structural alterations of the arterial wall in pathophysiological processes such as arterial hypertension, atheroma, or normal aging. The observed alterations are related to the three activities of the vascular smooth muscle cell, namely contractility, secretion of proteins from the extracellular matrix, and proliferation and migration. In arterial hypertension, vascular smooth muscle cells are functionally more contracted and structurally hypertrophic, and more collagen is secreted than under normal conditions. Similar structural changes are observed in the normal aging process. With respect to vascular smooth muscle cells, atheroma is characterized by their subintimal migration and proliferation, and by excessive excretion of collagen associated with other phenotypic modifications that are expressed in their regression to a myofibroblastic state. Regardless of the pathophysiological context, these phenotypic modifications of the vascular smooth muscle cell are always linked to an activation of the phosphoinositol pathways and to calcium accumulation. The activation of the phosphoinositol pathways seems to be a common feature of the different types of arterial hypertension. This activation can be associated with an increase in vasoactive peptides such as angiotensin II, vasopressin, or endothelin as in the secondary types of hypertension or directly related to an increase in vasoconstriction; or, as an exception, it can be spontaneously active in vivo and in vitro, as in the model of cultured vascular smooth muscle cells of the spontaneously hypertensive rat (SHR).(ABSTRACT TRUNCATED AT 250 WORDS)

The hemodynamic and myocardial effects of chronic treatment (5 months) with converting enzyme inhibitor (CEI, enalapril, group T1, n = 10) or a beta 1 blocker (atenolol, group T2, n = 13) of normotensive mature male Wistar rats were studied and compared to untreated younger rats (group C0, 11 months old, n = 14) and to untreated age-matched controls (group C1, 16 months old, n = 14). Heart rate was significantly decreased in T2 rats but unchanged in the T1 group. There was a significant decrease in systolic and diastolic arterial blood pressure (BP) in both treated groups (systolic T1:90 +/- 17, T2: 87 +/- 10, vs. C1: 102 +/- 11 mm Hg, and diastolic T1: 60 +/- 17, T2: 59 +/- 9, vs. C1: 73 +/- 13 mm Hg). Cardiac output was unchanged by therapy but stroke volume was significantly increased in the T2 group (0.24 +/- 0.03 vs. 0.19 +/- 0.04 cm3 in C1). Total peripheral resistance was significantly decreased in T1 and T2 groups. Left ventricular weight to body weight ratio (LVW/BW) was significantly decreased in T1 (1.74 +/- 0.11 vs. 2.02 +/- 0.22 in C1) but unchanged in T2 (1.90 +/- 0.15 vs. 2.02 +/- 0.22). The left ventricular passive pressure-volume curve was significantly rightward shifted by beta-blocker therapy (T2) but unchanged by CEI (T1). This corresponds to a decrease of chamber stiffness k in the T2 group compared to C1 and T1 groups. A significant fibrosis was seen in group T2 (mean collagen fiber thickness of 3.03 +/- 0.24 vs. 2.8 +/- 0.15 micron) but not in the T1 group (2.94 +/- 0.14 vs. 2.8 +/- 0.15 micron). Thus, despite the similar decrease in BP induced by the CEI and the beta blocker, the cardiac effects of the two drugs were opposite: CEI decreased LVW/BW with no change in the left ventricular pressure-volume curve and myocardial fibrosis whereas beta blocker did not change the LVW/BW, but induced a left ventricular dilation associated with a decrease in chamber stiffness and an increase in subendocardial fibrosis.

The effect of hypertension and of therapy by converting enzyme inhibitor (S 9490-3, perindopril) on the function and structure of large arteries has been studied in two-kidney, one-clip Goldblatt hypertensive rats. After one month without treatment, clipped hypertensive rats (n = 24) and sham-operated rats (n = 24) were randomly allocated to treatment by S 9490, 1 mg/kg once a day (n = 24) or to placebo (n = 24) and pursued for 4 weeks. Hemodynamic parameters, including instantaneous pressure and aortic velocity measured by Döppler, were recorded under anesthesia at the end of the treatment period. Passive mechanical properties of carotid arteries were recorded in situ in the presence or the absence of smooth muscle cell activity (potassium cyanide poisoning). Morphological parameters of the aortic media, including medial thickness, nucleus density, and cross sectional area and relative density in proteins of interstitial matrix, were recorded by an automated morphometrical system. Hypertension was associated with an increase in characteristic impedance of the aorta and a decrease in compliance of the arterial system. Treatment with converting enzyme inhibitors completely reversed these in vivo markers of the rigidity of large arteries. Hypertension was associated with a shift of the passive pressure-volume relation in the carotid. Treatment with converting enzyme inhibitors normalized the carotid pressure-volume relation, whereas poisoning smooth muscle cells induced a disappearance of the curve differences between hypertensive and normotensive animals. Morphometric analysis of aortic walls permits us to report this functional change to structural modification of the arterial wall. Aortic media thickness was increased by hypertension; this phenomenon was reversed by treatment. Modification of aortic thickness was due to hypertrophy of smooth muscle cells with parallel modifications of absolute amount of collagen, whereas absolute amount of elastin did not change in this early phase of renovascular hypertension in young rats. Treatment with converting enzyme inhibitors reversed the thickness of aortic media without regression of the increase in absolute amount of collagen content whereas absolute amount of elastin content did not change.

The effects of converting enzyme inhibition on the cardiac mass, isomyosins polymorphism, and collagen network in the left ventricle have been studied in renovascular, hypertensive, spontaneously hypertensive, and normotensive rats. The isoenzyme profile of left ventricular myosins was used as an indirect marker of the intrinsic property of contractility, whereas the collagen network, measured by a morphometric method, represented an indirect structural marker of the arrhythmogenic risk. One-clip, two-kidney renovascular hypertension was associated with cardiac hypertrophy, a shift in the isomyosin profile, and accumulation of collagen within the left ventricular myocardium. In this renin-angiotensin-dependent model, one month of treatment with converting enzyme inhibitor normalized blood pressure and consistently reversed cardiac hypertrophy and the isomyosin profile. Converting enzyme inhibitor treatment of 12-week-old spontaneously hypertensive rats for three months significantly decreased blood pressure but did not completely normalize it. The increase in cardiac mass observed in spontaneously hypertensive rats was not reversed by this short treatment. Nevertheless, the percentage of the V1 form of myosin increased slightly after treatment, and the collagen content of the left ventricle was considerably decreased. Converting enzyme inhibition did not decrease blood pressure in DOCA-salt hypertension, and no changes were observed in cardiac hypertrophy, isomyosin profile, or the collagen network. The cardiac hypertrophy that occurs with aging in normotensive rats was associated with a significant shift in isomyosin profile and a large accumulation of collagen. Thus, aging mimics several of the quantitative and qualitative changes in the left ventricular protein profile observed in hypertension. In young normotensive rats, converting enzyme inhibition significantly decreased blood pressure and left ventricular mass, increased the percentage of V1 isomyosin, and prevented the accumulation of collagen. In one-year-old normotensive rats, treatment for six months with converting enzyme inhibitor decreased blood pressure, decreased cardiac mass, and prevented the accumulation of collagen; the isomyosin profile was not modified. Converting enzyme inhibition, by acting on cardiac afterload, can bring about quantitative and qualitative changes in the cardiac proteins of both hypertensive and normotensive rats.

To explain how converting enzyme inhibition could improve the prognosis in cardiac insufficiency, the effect of converting enzyme inhibition (CEI) by S9490-3 (Perindopril) treatment for 2 months (treated infarctions, n = 18) on hormonal plasma variables and the quantitative and qualitative changes in myocardium were studied in an experimental model of left ventricular infarction in rats (untreated infarctions, n = 18) and compared to a sham-operated control group (n = 15). Induction of myocardial infarction was associated with a transient decrease in blood pressure. CEI treatment maintained a lower blood pressure throughout the experimental period. Plasma renin concentration was not significantly increased in the untreated infarct group (155.4 +/- 136.7 ng AI/ml/hr) as compared to the sham-operated group (47.6 +/- 15.9 ng AI/ml/hr). Plasma aldosterone did not change in the three experimental groups. The plasma level of immunoreactive atrial natriuretic factor increased in the untreated infarct group (185 +/- 245 pg/ml) as compared with the control group (76 +/- 40 pg/ml) and was normalized by CEI (66 +/- 60 pg/ml). Body weight was slightly decreased in both treated and untreated infarct groups, whereas the heart weight was significantly increased in the untreated group (1,540 +/- 310 mg) and normalized by treatment (1,145 +/- 180 mg) as compared with sham-operated controls (1,071 +/- 80 mg). The combined atria and right ventricular mass was significantly increased in the untreated infarct group (660 +/- 210 mg) and decreased by treatment (443 +/- 106 mg) but was not completely normalized (controls, 343 +/- 40 mg). Left ventricular isomyosin profiles were modified by myocardial infarction as compared with controls: V1 form decreased from 62.4 +/- 9.4% in the sham-operated group to 41.6 +/- 13.4% in the infarct group, and the V3 form increased from 13.0 +/- 4.7% in sham-operated animals to 27.4 +/- 11.8% in untreated infarct animals. CEI treatment partially, but significantly, reversed this modification of the isomyosin profile (V1, 53.0 +/- 14.4%; V3, 17.5 +/- 8.0%). Volume density of collagen was significantly increased in the untreated infarct rats (4.14 +/- 0.81% versus 2.68 +/- 0.49% in controls), and this was reversed by treatment (2.95 +/- 0.66%). Messenger RNA encoding for atrial natriuretic factor, measured by dot blot hybridization, was significantly increased in both the atria and the ventricles in the untreated infarct group, and treatment by CEI partially reversed this increase. Thus, myocardial infarction profoundly modified several variables of peripheral circulation and quantitative and qualitative myocardial protein expression.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of hypertension and its treatment on the function and structure of arteries have been extensively studied, but no data are yet available on the effect of hypotensive drugs on the large arteries in normotensive rats. Two groups of 11-month-old normotensive breeder male rats were treated for 5 months, one with an angiotensin converting enzyme (ACE) inhibitor (MK-421, 2 mg/kg per day; n = 10) and the other with dihydralazine (15 mg/kg per day; n = 8). A group of 14 rats served as controls. Blood pressure was recorded every 14 days by the tail-cuff method. At the end of the treatment period (5 months), the rats were killed under anaesthesia and the descending thoracic aorta was removed and fixed. The different components of the aorta were assessed by automated morphometric image analysis after specific coloration of elastin (orceine), collagen (sirius red) and nuclei (haematoxylin after periodic acid oxidation. Both the ACE inhibitor and dihydralazine caused similar decreases in systolic blood pressure (SBP) compared with controls. This hypotensive effect was associated with a reduction in medial thickness, from 120 +/- 15 microns in controls to 104 +/- 9 microns in ACE inhibitor-treated and 103 +/- 10 microns in dihydralazine-treated normotensive rats. Elastin density significantly increased in the two treatment groups but the greatest increase was in the dihydralazine-treated group (P less than 0.001). Elastin fibre thickness increased significantly in the dihydralazine-treated group only. Collagen density was not significantly modified by either treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of chronic therapy, using an angiotensin converting enzyme (ACE) inhibitor (S9490-3 perindopril, 1 mg/kg), on the mechanical and structural properties of large arteries were studied in two-kidney, one clip (2K, 1C) Goldblatt hypertensive and spontaneously hypertensive rats (SHR) compared with matched normotensive Wistar and Wistar-Kyoto (WKY) animals. The treatment was carried out for 1 month in Goldblatt-hypertensive rats and 3 months in SHR, i.e. for 1 month after blood pressure was normalized. At the end of the treatment period the passive mechanical properties of the isolated carotid artery were measured in situ. Carotid compliance was calculated from the pressure-volume relationship between 50 and 250 mmHg. Morphological parameters of the aortic wall, including medial thickness, nucleus density and elastin and collagen content, were recorded by an automated morphometric system. Renal and essential hypertension were associated with a shift of the passive pressure-volume relationship in the carotid, corresponding to a decrease in arterial compliance. The passive mechanical properties of the carotid were normalized by ACE inhibitor treatment in renovascular hypertensive rats but remained unchanged in chronically treated SHR. The ACE inhibitor completely reversed the medial hypertrophy in Goldblatt-hypertensive rats but the reversal of medial hypertrophy was incomplete in SHR. The elastin to collagen ratio in the aortic media was significantly increased by 3 months of treatment with the ACE inhibitor in the SHR and WKY groups but remained unchanged in the Goldblatt-hypertensive and Wistar rats treated for 1 month.

The effects of renovascular hypertension and of its treatment with perindopril, a converting enzyme inhibitor, on the structure and function of large arteries were studied on 2 kidneys, 1 clip Goldblatt rats. One month after surgery, the animals rendered hypertensive (n = 24) and those who had had a blank operation (n = 24) were divided into two groups receiving either perindopril 1 mg/kg/day or distilled water during 4 week At the end of treatment haemodynamic values, including arterial pressure and instant blood flow at Doppler velocimetry, were measured in anaesthesized rats. The mechanical properties of the carotid artery were studied by in situ measurement of carotid compliance in response to imposed pressures. Finally, morphometric parameters of the thoracic aorta, including thickness of the media, density of elastin, collagen and nuclei and nuclear surface area, were studied by means of an automatized image analysis system. Hypertension was associated with a characteristic increase in aortic impedance (14,479 +/- 5,171 vs 9,022 +/- 4,071 dyn.sec/cm5; p less than 0.01) and a decrease in systemic arterial compliance (2.41 +/- 0.96 vs 3.92 +/- 1.15 x 10-3 ml/mmHg; p less than 0.05) and carotid compliance (6.31 +/- 1.85 vs 3.8 +/- 3.4 X 10-2 mm3/mmHg; p less than 0.05). Treatment with perindopril normalized the systolic and diastolic pressures and completely reversed the artery rigidity markers. Our morphometric analysis of the aortic wall enabled us to relate these functional changes to structural changes in vascular wall.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of converting enzyme inhibitor have been studied on the functions and structure of the arterial wall, and in both models of renovascular and spontaneously hypertensive rats. The arterial wall functions have been studied in terms of active vasomotricity, passive elasticity and transparietal convexion transport of proteins. Whatever the model used, hypertension was associated with significant changes in arterial wall functions: increase in smooth muscle cell tone, decrease in compliance, and decrease in transparietal filtration rate of proteins. These modifications could be reported to the increase in thickness of the media secondary to hypertrophy and hyperplasia of smooth muscle cell and to the increase in collagen network density. The effects of converting enzyme inhibitors on these phenomenons could depend on the experimental model used and the duration of treatment. In renovascular hypertensive animals, one month of treatment with perindopril normalized blood pressure and active compliance of the arterial wall. This functional benefit could be easily reported to the complete regression of the smooth muscle cell hypertrophy observed after treatment. By contrast, the increase in collagen network density was not regressed by treatment. By contrast, the increase in collagen network density was not regressed by treatment. By contrast, the increase in collagen network density was not regressed by treatment with trandolapril and thus the transparietal filtration rate of proteins remained decreased. In spontaneously hypertensive rats a perindopril treatment of long duration (3 months) partially regressed the functional change in compliance. This effect, once again, could be easily reported to the partial regression of medial hypertrophy which was associated, in this experimental condition, with a significant decrease in collagen network density.

It has been suggested that complex ventricular arrhythmias commonly occur in hypertensive patients with left ventricular hypertrophy. We have previously demonstrated that coronary artery ligation in anesthetized spontaneously hypertensive rats (SHR) and their normotensive controls (WKY) resulted in a significantly increased incidence and duration of ventricular fibrillation in SHR compared with WKY. The object of the present study was to characterize the structural and electrophysiological abnormalities in hypertrophied hearts, associated with the occurrence of arrhythmias. We used a double tissue bath in which a ventricular strip was exposed simultaneously to normal and to altered conditions (low pH, hypoxia and high potassium). Electrical activity recorded using standard micro-electrode techniques showed the occurrence of arrhythmias in all preparations and the development of major alterations in conduction (a conduction block appeared at 11 +/- 1 mn in SHR vs 16 +/- 1 mn in WKY, p less than 0.05), and maximal upstroke velocity (Vmax values before and 3 mn after the beginning of ischemia were 229 +/- 12 to 46 +/- 7 v/s for the SHR and 227 +/- 10 to 106 +/- 12 v/s for the WKY; p less than 0.001). These changes were associated in hypertrophied ventricles with a marked sub-endocardial collagen fibrosis as estimated by the use of automated image analysis (subendocardial collagen density = 4.39 +/- 0.34 p. 100 in SHR vs 1.66 +/- 0.15 p. 100 in WKY; p less than 0.001). Action potential duration measured using conventional glass micro-electrodes in a single chamber tissue bath revealed a highly significant difference (p less than 0.001) in APD 90 p. 100 of papillary muscles between SHR (114.7 +/- 2.8 ms) and WKY (76.9 +/- 1.7 ms). The addition of tetra-ethylammonium to block potassium channels induced triggered activity arising from early afterdepolarizations only in muscles hypertrophied SHR hearts.(ABSTRACT TRUNCATED AT 250 WORDS)