Inside-out and outside-in organotypic normal human skin culture: JAK-STAT pathway is activated after pro-inflammatory psoriatic cytokine exposure.

The Janus kinases-signal transducers and activators of transcription (JAK-STAT) signalling pathway are a pleiotropic cascade that involves ligands such as cytokines, hormones, and growth factors. Among cytokines, interleukin (IL)-17, IL-22, IL-23, and tumour necrosis factor (TNF)-alpha play a pivotal role in psoriasis. We aimed at investigating in an organotypic experimental model of normal human skin (n = 7 women between 20-40 years old, non-smokers) the early, direct, and specific effects of IL-17, IL-22, IL-23, TNF-alpha and a combination of the four cytokines (Mix) on the JAK-STAT/pathway. The expression of the psoriatic marker keratin (K) 17 was analyzed by immunofluorescence and molecular techniques after exposure to IL-23 or Mix. The Mix elicited a strong K17 up-regulation in keratinocytes at 72 h, reinforcing the hypothesis of a synergistic effect of different cytokines. High levels of JAK1 and STAT3 activation were detected, suggesting the involvement of JAK1/STAT3 pathway in the upregulation of K17. As the present study in an organotypic model of human skin reports a variable expression of JAK-STAT upon different cytokine stimuli and most of the JAK inhibitors for the psoriasis treatment have proven to have a clinical efficacy, these observations have a relevance to better understand the mechanisms of JAK-inhibitors in the skin.

Relevance of in vitro 3-D skin models in dissecting cytokine contribution to psoriasis pathogenesis.

Psoriasis is a chronic skin disease characterized by the activation of various T cell subsets secreting IFNγ, IL-17, and IL-22, dendritic cells producing TNFα and IFNα, and other immune cells including neutrophils and mast cells. Keratinocytes respond to different cytokine signals orchestrating innate and adaptive immune responses. In vitro studies sought to clarify the cytokine effects on keratinocytes in order to evaluate the centrality of these mediators in psoriasis pathogenesis. The aim of this review is to highlight the relevance of this peculiar in vitro approach in investigating cytokine effects on skin or multilayered epidermis. Particularly, we reported keynfindings supporting the cytokine role in psoriasis pathogenesis.

An innovative three-dimensional model of normal human skin to study the proinflammatory psoriatic effects of tumor necrosis factor-alpha and interleukin-17.

BACKGROUND: Among all cytokines involved in the pathogenesis and in the progression of psoriasis, Tumor Necrosis Factor (TNF)-alpha and interleukin (IL)-17 play a pivotal role. OBJECTIVE: The aim of the present study was to mimic a psoriatic microenvironment and to investigate the early effects of TNF-alpha and IL-17 in a three-dimensional model of organotypic normal human skin. METHODS: Human skin explants were obtained from plastic aesthetic surgery of healthy young women 20-40years old (n=7). The study was approved by the Institutional Review Board and written informed consent was obtained from all subjects. Bioptic fragments were cultured at the air-liquid interface overnight in a Transwell system and further divided before adding either 50ng/ml IL-17 or 100ng/ml TNF-alpha or a combination of both cytokines. For each subject, a control sample was cultured without any cytokine. Samples were harvested 24 or 48h after cytokine incubation. At both time points and for all cytokine treatments a bioptic fragment obtained from each patient was processed. Epidermal proliferation, expressions of terminal differentiation (keratin 10, K10, and 14, K14) and of intercellular adhesion (occludin for tight junctions and E-cadherin for adherens junctions) biomarkers were investigated by indirect immunofluorescence. RESULTS: IL-17 and TNF-alpha induced an early and statistically significant inhibition of keratinocyte proliferation (more than 80% compared with their respective controls). At 24h, the combination of both cytokines did not further reduce cell proliferation. Starting from 24h of incubation, a non-continuous occludin expression in the granular layer was observed after both IL-17 and TNF-alpha exposure. Immunolabelling for E-cadherin in adherens junctions, for K10 in the suprabasal layers, and for K14 in the basal layer was similar in all experimental groups and unaffected after cytokine treatment. CONCLUSIONS: These results suggest that in this experimental model IL-17 and TNF-alpha induced an early alteration of the homeostasis of the inner proliferative layer and of the upper granular layer, as shown by cell proliferation inhibition and occludin expression.

Alendronate impairs epithelial adhesion, differentiation and proliferation in human oral mucosa.

OBJECTIVE: This study aimed at evaluating from a morphological point of view the effects of alendronate (ALN), a widely used nitrogen-containing bisphosphonate for the chronic treatment of osteoporosis, on the oral epithelium of healthy keratinized human oral mucosa. Bisphosphonate-related osteonecrosis of the jaw is a well-known severe consequence, but the effects during chronic therapy on the oral soft tissues are still matter of debate. MATERIALS AND METHODS: Six women over 60 year-old undergoing treatment of osteoporosis with 70 mg per week of oral ALN (lasting at least 2 years) were recruited and compared with a gender and age-matched group (n = 6). Proliferation, apoptosis, intercellular adhesion and terminal differentiation (TD) were investigated by immunofluorescence. In parallel, ultrastructural analysis was carried out. RESULTS: By immunofluorescence, a statistically significant decrease in keratinocyte proliferation was detected in the oral epithelium of the ALN group without any sign of apoptosis, but accompanied by a reduction in desmoglein 1 and keratin 10 expressions. In the uppermost layers of the oral epithelium of the ALN group, thin desmosomes were visible by transmission electron microscopy. CONCLUSION: Our results show that epithelial adhesion, TD and proliferation are affected by ALN therapeutic doses in clinically healthy human oral mucosa.

Skin immunosenescence: decreased receptor for activated C kinase-1 expression correlates with defective tumour necrosis factor-alpha production in epidermal cells.

BACKGROUND: Skin immunosenescence accounts for increased susceptibility in the elderly to cutaneous infections and malignancies, and decreased contact hypersensitivity and response to vaccination. We have recently shown in immune cells that decreased expression of the receptor for activated C kinase (RACK)-1 underlies defective protein kinase C (PKC) activation and functional immune impairment with ageing. OBJECTIVES: This study was designed to determine if an age-related decline in skin RACK-1 expression was present and whether it correlated with defective tumour necrosis factor (TNF)-alpha production. METHODS: PKC isoforms and RACK-1 expression were evaluated by Western blot analysis and by immunofluorescence in skin obtained from Sprague-Dawley rats of different ages. TNF-alpha release by epidermal cells induced by lipopolysaccharide, 12-O-tetradecanoyl-phorbol-13-acetate and the contact allergen dinitrochlorobenzene was assessed by the L929 biological assay. RESULTS: Skin obtained from old rats (> 18 months) showed decreased RACK-1 immunoreactivity if compared with young rats (< 3 months). RACK-1 preferentially interacts with PKC beta. Despite a similar total skin content of this isoform, the reduced expression of RACK-1 was associated with a decreased translocation of PKC beta in the membrane compartment. The defective PKC beta translocation associated with ageing correlated with decreased TNF-alpha release from epidermal cells following treatment with different inflammatory stimuli. CONCLUSIONS: Overall, we demonstrated for the first time a decrease in RACK-1 expression, defective PKC beta translocation and reduced TNF-alpha release in epidermal cells with ageing. These alterations might be mechanistically significant, and provide a new understanding of the consequences of ageing on skin immunology.

Effect of a topical treatment in organotypic culture of human breast skin after exposure to gamma-rays.

The early radiation of epidermal reactions can lead to healing of the lesion or radiation necrosis. There is no general agreement for either the prevention and/or treatment of radiation skin response, also as little is known about the immediate phases of this phenomenon. We investigated the early effects exerted by Healing and Wound Emulsion (HWE) on human skin response after ionizing radiation. Epidermal morphology, Heat Shock Protein (HSP) 70, and Transforming Growth Factor-beta1 (TGF-beta1) gene expression were investigated in organotypic human skin cultures undergoing a double dose of gamma-rays (2 Gy). HSP70 gene expression tended to be induced in the HWE group 6 hours after cream administration and was significantly up-regulated after 48 hours, when epidermal morphological alterations were evident. TGF-beta1 seems not affected in cream treated samples. HWE may stimulate skin to mount an early defensive response against damage induced by gamma rays.

Early epidermal response after a single dose of gamma-rays in organotypic culture of human breast skin.

BACKGROUND: Skin reaction is the most common side-effect of radiation therapy. Radiation-induced dermal fibrosis has been characterized histologically, but little is known about the epidermis overlying fibronecrotic lesions. OBJECTIVES: To characterize the epidermal response 24 h after a single clinically relevant dose of gamma-rays in cultured human breast skin. METHODS: Biopsies obtained from cosmetic surgery (n = 7) were placed epidermis upwards in a Transwell system, and were exposed to a single dose of gamma-irradiation (2 Gy). A parallel set of nonirradiated skin fragments was incubated under the same conditions. Both irradiated and nonirradiated fragments were harvested 24 h after irradiation and processed for light microscopy and molecular biology analysis. A quantitative analysis of cell proliferation was performed after 5-bromo-2'-deoxyuridine incorporation. Cytokeratin 10 (CK10) and desmocollin 1 (Dsc1) expression was evaluated by immunofluorescence. Dsc1 and transforming growth factor (TGF)-beta1 gene expression was measured by reverse transcriptase-polymerase chain reaction analysis. RESULTS: The mean percentage inhibition of epidermal proliferation in irradiated samples was 53.7% (P < 0.01, paired Student's t-test). The inhibition of cell proliferation was significant in five of seven samples (P < 0.05, unpaired Student's t-test). Normal cell architecture was found in irradiated samples. Throughout the epithelial compartment, the distribution patterns of CK10 and Dsc1 were comparable in nonirradiated and irradiated fragments. Condensation of CK10 filaments suggested a cytoskeletal rearrangement in irradiated samples. Dsc1 and TGF-beta1 mRNA levels were, respectively, reduced and unmodified 24 h after irradiation. CONCLUSIONS: A perturbation of epidermal homeostasis occurs as early as 24 h after a single dose of gamma-rays. Our immunofluorescence observations indicate that keratinocyte terminal differentiation is not yet affected at the protein level 24 h after exposure to gamma-rays. The lack of an inverse relationship between TGF-beta1 gene expression and epidermal proliferation, together with decreased Dsc1 gene expression, may represent the early molecular basis for the development of the late effects of radiotherapy observed many months/years after radiotherapy. Our findings set the stage for further investigation of the best time to begin topical treatment at the start of radiation therapy.

Pattern of expression of c-Myc, Max and Bin1 in human anagen hair follicles.

BACKGROUND: We recently reported the presence of c-Myc immunoreactivity in two distinct regions of the inner root sheath (IRS) of human anagen hair follicles; they corresponded to the regions where keratinocytes of Henle's and Huxley's layers enter the terminal differentiation phase that will lead to their exfoliation in the pilary canal. These regions were denoted lower (LR) ring and upper ring (UR). OBJECTIVES: To extend these observations to other genes connected to c-Myc and specifically to Max and Bin1. Max is the best known heterodimeric partner of c-Myc, interacting with its C-terminal domain, and Bin1 is an adaptor protein interacting with its N-terminal domain. METHODS: Human anagen hair follicles were processed for c-Myc, Max and Bin1 immunohistochemistry and immunofluorescence. The presence of different isoforms of Bin1 was evaluated by Western blot analysis. RESULTS: Analysis of sections cut in several planes, including tangential, demonstrated the presence of a third ring of c-Myc-positive cells (intermediate ring; IR) in the cuticle of the IRS corresponding to the region where this thin layer undergoes keratinization. Max immunoreactivity was observed in the three layers of the IRS starting in the lower bulbar region and ending in each of them at the level of the corresponding c-Myc-positive ring. Bin1 immunoreactivity was clearly distinguished only in Huxley's layer and in the cuticle, starting in some cells below the UR and terminating at the level of the latter. The companion layer of the outer root sheath was also labelled up to the infundibular region. Max and Bin1 immunostaining were less consistently observed in other skin adnexae and in the epidermis. CONCLUSIONS: The results indicate that the asynchronous differentiation along the axis of the hair follicle of the different layers of the IRS and of the companion layer involves the expression of different genes that are interrelated in the so-called 'Myc network'. The specific localization of c-Myc in the IRS only at the level of the discrete and limited regions of the three rings appears to be the hallmark of the switch from differentiation to terminal differentiation/cell deletion.

c-Myc expression in human anagen hair follicles.

The hair follicle represents a very attractive organ system for studying the precise balance between cell proliferation, growth, differentiation, and death of cells, because it periodically and regularly regenerates, retaining its morphogenetic signals throughout its life. One of the most intriguing oncogenes which is able to induce both cell growth and apoptosis, depending upon the environmental conditions, is c-myc. The aim of the present study was to investigate its presence and localization in human hair follicles by immunohistochemistry and immunofluorescence. Our observations demonstrated the consistent presence of two clusters of c-Myc-expressing cells in anagen follicles, located in two annular regions of the inner root sheath, at the border between cells characterized by putative trichohyalin granules and cells which are keratinized. The lower group belongs to Henle's layer, while the upper group belongs to Huxley's layer. c-Myc oncoprotein seems to favour apoptosis/differentiation and may be a marker for terminal differentiation of trichocytes, at least in the inner root sheath. Our findings agree with the interpretation that the complex morphology of the hair follicle reflects its complex function; the extrusion of a highly organized multicellular structure, the hair shaft, driven by another highly organized multicellular structure, the inner root sheath.

Dual effects of the antioxidant agents probucol and carvedilol on proliferative and fatty lesions in hypercholesterolemic rabbits.

The in vivo direct antiatherogenic activity of the antioxidant probucol (200 mg/kg per day) or the beta-blocker with antioxidant properties carvedilol (10 and 20 mg/kg per day) was tested in the same animal in two different types of atherosclerotic lesion (proliferative and fatty lesions) induced in cholesterol-fed rabbits (1%). Drugs were given daily mixed with standard diet for 8 weeks; body weight and plasma lipid profile were not different among groups throughout the study. Aortic fatty lesions were induced by cholesterol feeding (n = 25 in each group) and their extent expressed as % of aorta inner surface covered by plaques was significantly reduced by both drugs (28.2+/-9.6%, P <0.05, 19.9+/-6.2%, P <0.01 for low- and high-dose carvedilol, respectively; 22.3+/-7.6%, P <0.01 for probucol, versus 41.6+/-10.7% in control rabbits). Proliferative lesions were obtained by positioning a hollow silastic collar around one carotid artery 6 weeks after dietary and drug treatments started (n = 5 in each group). The neointimal formation, mostly composed by myocytes, was determined by measuring cross-sectional thickness ratio of intimal (I) and medial (M) tissue of fixed arteries. In untreated animals, collared arteries resulted in a significant neointimal cell accumulation compared to the sham (1.10+/-0.14 versus 0.02+/-0.01) without change in medial thickness. I/M ratio was reduced by about 50% in animals treated with probucol (0.51+/-0.1) and carvedilol (0.66+/-0.21 and 0.52+/-0.1 in the low- and high-dose group, respectively). Total plasma TBARS were more than 50% lower in both probucol- and high-dose carvedilol-treated rabbits. Results show that pharmacological pretreatment with antioxidants directly inhibits early atherogenic processes, representing a potentially useful approach in the prevention of atherosclerosis.

Effect of lercanidipine and its (R)-enantiomer on atherosclerotic lesions induced in hypercholesterolemic rabbits.

The in vivo antiatherogenic activity of the calcium antagonist lercanidipine and its (R)-enantiomer was investigated in two different types of atherosclerotic lesions (hyperplastic and fatty-streak lesions) in rabbits. Lercanidipine (0.3, 1, and 3 mg kg(-1) week(-1)) as well as its (R)-enantiomer at 3 mg kg(-1) week(-1) were given by subcutaneous injection for 10 weeks to White New Zealand rabbits, with cholesterol feeding beginning at week 2. The hyperplastic lesion was obtained by positioning a hollow silastic collar around one carotid artery, while aortic fatty streak lesions were induced by cholesterol feeding. In untreated animals (n=5), 14 days after collar positioning an intimal hyperplasia was clearly detectable: the arteries without collar showed a intima/media (I/M) ratio of 0.03+/-0.02, whereas in carotids with a collar the ratio was 2+/-0.42. In lercanidipine-treated animals a significant and dose-dependent effect on intimal hyperplasia was observed. I/M ratios were 0.73+/-0.4, 0.42+/-0.1, 0.32+/-0.1 for 0.3, 1, and 3 mg kg(-1) week(-1), respectively (P<0.05). The lercanidipine enantiomer (3 mg kg(-1) week(-1)) was as effective as the racemate (0.41+/-0.11). Proliferation of smooth muscle cells, assessed by incorporation of BrdU into DNA, was reduced by about 50%, 70%, 85%, and 80% by lercanidipine (0.3, 1, and 3 mg kg(-1) week(-1)) and its (R)-enantiomer, respectively. The area of fatty-streaks in the aorta (n = 11-15) was significantly reduced by lercanidipine (3 mg kg(-1) week(-1), 16% vs 27%, P<0.05), a trend was observed also with lower doses. When different segments of the aorta were considered (arch, thoracic, abdominal) a significant and dose-dependent effect in the thoracic and abdominal aorta was observed also at lower doses. The (R)-enantiomer was as effective as lercanidipine. These results suggest a direct antiatherosclerotic effect of lercanidipine, independent of modulation of risk factors such as hypercholesterolemia and/or hypertension as demonstrated by the absence of stereoselectivity.

Gene transfer into the carotid artery using an adventitial collar: comparison of the effectiveness of the plasmid-liposome complexes, retroviruses, pseudotyped retroviruses, and adenoviruses.

We studied the efficiency of plasmid/liposome complexes, Moloney murine leukemia virus-derived (MMLV) retroviruses, pseudotyped vesicular stomatitis virus protein-G (VSV-G)-containing retroviruses, and adenoviruses in delivering genes into the rabbit carotid artery using a silastic collar applied to the adventitia. This method was used for gene transfer because (a) it provides a gene delivery reservoir; (b) no intraluminal manipulations are performed; (c) installation of the collar induces arterial smooth muscle cell (SMC) proliferation and enhances retroviral gene transfer efficiency where target cell proliferation is required. The transfer of the beta-galactosidase (lacZ) marker gene to the adventitia and media occurred with all gene transfer systems. Adenoviruses also transferred the beta-galactosidase gene to some endothelial cells. After 5 days, adenoviral vectors produced the highest gene transfer efficiency with up to 10%+/-6% of cells showing beta-galactosidase activity. Pseudotyped VSV-G retroviruses were also effective in achieving gene transfer in 0.05%+/-0.03% of cells in the adventitia and media. Plasmid/liposome complexes and MMLV retroviruses infected 0.05%+/-0.03% and <0.01%+/-0.01% of cells, respectively. It is concluded that replication-deficient adenoviruses, VSV-G pseudotyped retroviruses, and plasmid/liposome complexes can be used for gene transfer to the arterial wall using the collar method. Because the endothelium remains anatomically present throughout the experiments, the model may be useful for the gene transfer studies involving diffusible or secreted gene products that primarily act on the endothelium. Effects on medial SMC and even endothelium can be achieved from the adventitial side, suggesting an alternative route for the delivery of therapeutically useful genes into the arterial wall.

Proapoptotic effect of atorvastatin on stimulated rabbit smooth muscle cells.

The in vitro and in vivo activity of atorvastatin and other 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase inhibitors (fluvastatin, pravastatin and simvastatin) has been investigated. Atorvastatin, fluvastatin, pravastatin and simvastatin caused a significant and dose-dependent (0.1-50 microM) reduction in cell multiplication of vascular smooth muscle cells (SMC) in cultures associated with the retardation of cycling cells in the G1 and G2/M compartments at 24 h, a phenomenon leading to apoptosis (programmed cell death) in several experimental in vitro models. The effects on the cell cycle resulted in a strong inhibition of cell proliferation at 48 h, followed by apoptosis when incubation was prolonged to 72 h as assessed by nuclei morphology and cytofluorimetric analysis of DNA. The apoptotic effect observed for the statins is completely prevented by the addition of exogenous mevalonate at a 100 microm concentration. in vivo SMC proliferation was stimulated by applying a silastic collar to the outside surface of carotid arteries in normocholesterolemic rabbits in the presence of an anatomically intact endothelium. The positioning of the collar promoted apoptosis in control vessels as assessed by Terminal Deoxynucleotidyl Transferase-dUTP-Biotin Nick-End Labeling (TUNEL) assay. The pre-treatment with 5 mg kg-1 per day of atorvastatin before collar insertion strongly increased the number of TUNEL-positive cells, suggesting a pro-apoptotic effect of HMGCoA reductase inhibitors also in vivo, even though cell DNA rearrangement still needs to be excluded. No apoptotic signal was detectable in sham operated arteries with no collar in either control or atorvastatin-treated rabbits. These data indicate that HMGCoA reductase inhibitors effect on the arterial wall may involve the modulation of both cell proliferation and programmed cell deaths supporting a possible role of statins in the prevention of early lesion and restenosis.

Direct antiatherogenic activity of isradipine and lacidipine on neointimal lesions induced by perivascular manipulation in rabbits.

The in vivo antiatherogenic activity of two calcium antagonists of the dihydropyridine class (isradipine and lacidipine) was investigated in a new experimental model. The proliferative lesion induced in the rabbit carotid artery was obtained by positioning a hollow silastic collar around the vessel. The neointimal formation was determined by measuring cross sectional thickness of intimal (I) and medial (M) tissue of fixed arteries obtained 14 days after collar placement. The effectiveness in inhibiting neointimal formation was assessed for isradipine (0.5, 1 and 4 mg kg-1 day-1) in normocholesterolemic (NC) animals and for lacidipine (1, 3, and 10 mg kg-1 day-1) in hypercholesterolemic (HC) rabbits. In NC control animals a neointimal formation was clearly detectable (I/M 0.53 +/- 0.18, n = 5). In isradipine-treated groups I/M ratios were significantly decreased (0.15 +/- 0.03, 0.12 +/- 0.02, 0.1 +/- 0.02 for the 0.5, 1 and 4 mg kg-1 day-1 doses respectively). In HC rabbits the administration of cholesterol 1% mixed with food and drug treatment started either 60 days before collar insertion (pretreated group, HC60) or on the same day (non pretreated group, HC15) of the collar placement. Only the pharmacological pretreatment was effective in reducing neointimal formation (0.47 +/- 0.02, 0.4 +/- 0.09, and 0.32 +/- 0.02 for dose 1, 3 and 10 mg kg-1 day-1 vs 1.1 +/- 0.14 in control animals). The inhibition of neointimal hyperplasia was much less evident in nonpretreated animals (0.7 +/- 0.15, 0.6 +/- 0.18 and 0.43 +/- 0.08 for dose 1, 3, and 10 mg kg-1 day-1 vs 0.72 +/- 0.2 in control animals). These results suggest a direct antiatherosclerotic effect of isradipine and lacidipine on neointimal hyperplasia induced in NC and HC pretreated rabbits independently of modulation of risk factors such as hypercholesterolemia and/or hypertension.

Non-lipid-related effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.

With the increasing knowledge of the pathogenesis of atherosclerosis, it appears that in the future the prevention of cardiovascular disease will involve not only risk factor correction, but also direct pharmacological control of processes occurring in the arterial wall. Among these, a pivotal role is played by smooth muscle cell (SMC) migration and proliferation, which, together with lipid deposition, are prominent features of atherogenesis and restenosis after angioplasty. Mevalonate and other intermediates of cholesterol synthesis (isoprenoids) are essential for cell growth, hence drugs affecting this metabolic pathway are potential antiatherosclerotic agents. Recently, we provided in vitro and in vivo evidence that fluvastatin, simvastatin and lovastatin, but not pravastatin, decrease SMC migration and proliferation dose dependently, independently of their hypocholesterolemic properties. The in vitro inhibition of cell migration and proliferation induced by simvastatin and fluvastatin (70-90% decrease) was prevented completely by the addition of mevalonate, and partially prevented by farnesol and geranylgeraniol (80%), confirming the specific role of isoprenoid metabolites in regulating these cellular events, probably through prenylated protein(s). The in vivo antiproliferative activity of fluvastatin on neointimal hyperplasia in normocholesterolemic rabbits was also prevented fully by the local delivery of mevalonate, by means of an Alzet pump. Fluvastatin and simvastatin also inhibited cholesterol esterification and deposition induced by acetylated LDL in cultured macrophages. This effect was fully prevented by the addition of mevalonate or geranylgeraniol. Taken together, these results suggest that, beyond their effects on plasma lipids, HMG-CoA reductase inhibitors exert a direct antiatherosclerotic effect on the arterial wall, probably through local inhibition of isoprenoid biosynthesis.

Effect of lacidipine on fatty and proliferative lesions induced in hypercholesterolaemic rabbits.

1. The in vivo antiatherogenic activity of the calcium antagonist, lacidipine, was investigated in two different types of atherosclerotic lesions (proliferative and fatty lesions) induced in rabbits. 2. The proliferative lesion was obtained by positioning a hollow silastic collar around one carotid artery, while aortic fatty lesions were induced by cholesterol feeding. Cholesterol (1%) and lacidipine (1, 3, and 10 mg kg-1) were given daily mixed with standard diet for 8 weeks to White New Zealand rabbits. The intimal hyperplasia (proliferative lesion) was induced 6 weeks after dietary and drug treatment started. 3. The neointimal formation was determined by measuring cross sectional thickness of intimal (I) and medial (M) tissue of fixed arteries. In untreated animals (n = 5), 14 days after collar positioning an intimal hyperplasia was clearly detectable: the arteries with no collar (sham) showed an I/M tissue ratio of 0.03 +/- 0.02, whereas in the carotid with collar the ratio was 0.62 +/- 0.12. In lacidipine-treated animals a significant and dose-dependent effect on proliferative lesions at all three doses tested, was observed. I/M ratios were 0.47 +/- 0.02, 0.40 +/- 0.09, 0.32 +/- 0.02 for doses 1, 3, and 10 mg kg-1 day-1, respectively (P < 0.05). 4. The fatty lesion extent was significantly reduced by lacidipine at the 10 mg kg-1 day-1 dose, although a trend was also observed with lower dosage. 5. These results suggest a direct antiatherosclerotic effect of lacidipine, independent of modulation of risk factors such as hypercholesterolaemia and/or hypertension. Furthermore, the proliferative lesions are apparently more sensitive to lacidipine than are lipid-rich lesions.

Recombinant apolipoprotein A-IMilano dimer inhibits carotid intimal thickening induced by perivascular manipulation in rabbits.

Apolipoprotein A-IMilano (apoA-IM), a natural variant of apolipoprotein A-I (apoA-I), confers to the carriers a significant protection against vascular disease. The antiatherogenic activity of a recombinant disulfide-linked apoA-IM dimer (rA-IM/A-IM) was analyzed in vivo by evaluating its effect on neointimal formation induced by periarterial manipulation in 1% cholesterol-fed rabbits. A flexible collar was applied around the carotid artery 21 days after the beginning of the dietary regimen, and animals were killed 10 days later. Rabbits were injected five times with reconstituted high-density lipoprotein containing egg phosphatidylcholine (EPC) and rA-IM/A-IM (119 mg EPC + 40 mg protein per dose) or with EPC liposomes (119 mg EPC per dose) beginning either 5 days before or at the day of collar positioning. Neither treatment affected plasma cholesterol levels. A significant intimal thickening was observed in control animals; the intima-to-media (I/M) ratio was 0.63 +/- 0.11 versus 0.03 +/- 0.05 for the sham-operated contralateral arteries. Neointimal formation was markedly inhibited in animals pretreated with rA-IM/A-IM before lesion induction (I/M, 0.26 +/- 0.19) but not in those in which treatment began the day of collar insertion (I/M, 0.74 +/- 0.14). EPC liposomes did not affect neointimal formation (I/M, 0.50 +/- 0.14 and 0.51 +/- 0.07 in the two treatment groups). Proliferation of smooth muscle cells, assessed by direct incorporation of bromo-2'-deoxyuridine (BrdU) into replicating DNA, was reduced by approximately 30% and 75% in the intimal and medial tissues of rA-IM/A-IM-pretreated rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of isoprenoid biosynthesis and arterial smooth-muscle cell proliferation.

Recently, we provided in vitro and in vivo evidence that several vastatins with different potencies decrease arterial smooth-muscle cell (SMC) proliferation independently of their hypocholesterolemic properties. In this study, the in vivo dose-dependent antiproliferative activity of fluvastatin on neointimal formation induced by the insertion of a collar around one carotid artery was investigated in normocholesterolemic rabbits (five animals per treatment group). Intraperitoneal fluvastatin treatment progressively inhibited intimal to medial tissue ratios (I/M) by 5, 48, and 64% versus controls at doses of 3, 5, or 10 mg/kg/day, respectively. Local arterial delivery by an Alzet pump of mevalonate (8 mg/kg/day) at the site of collar placement fully prevented a fluvastatin (5 mg/kg/day) inhibitory effect on both I/M and SMC proliferation, as assessed by direct incorporation of bromodeoxyuridine (BrdU) into replicating DNA. The results suggest that vastatins exert a direct antiproliferative effect on intimal myocytes beyond their effects on plasma lipids, probably through local inhibition of isoprenoid biosynthesis.

Effect of lacidipine on the carotid intimal hyperplasia induced by cuff injury.

The in vivo antiatherogenic activity of the calcium antagonist lacidipine was investigated in arterial hyperplasia induced by perivascular manipulation of hypercholesterolemic carotid rabbits. This was accomplished by positioning a hollow silastic collar around one carotid, which within a few days induces an atherosclerotic lesion (proliferative lesion) showing biochemical and morphologic changes similar to those of early human atherosclerosis: the contralateral carotid, with no collar, served as control in the same animal. The effect of lacidipine was also investigated in aortic atherosclerotic lesions (fatty lesions) induced by hypercholesterolemia mixed with either cholesterol (1%) and lacidipine (3 mg/kg/day) or cholesterol (1%) alone for 8 weeks. Hypercholesterolemic New Zealand White rabbits were fed daily a standard diet. Intimal hyperplasia was mechanically induced in one carotid artery of each rabbit 6 weeks after dietary and drug treatment started. Neointimal formation was followed by measuring by light microscopy the cross-sectional thickness of intimal (I) and medial (M) tissue of fixed arteries. In positive control animals receiving dietary cholesterol only (n = 10), by 14 d after collar positioning the process of intimal hyperplasia was significantly pronounced. The control arteries showed an I:M tissue ratio of 0.03 +/- 0.02, whereas in the carotid with collar the ratio was 0.56 +/- 0.11. In the animals receiving lacidipine, neointimal formation was significantly lower [I:M tissue ratio 0.32 +/- 0.1 (n = 10), about 60% of positive controls]. Measurement of the percent area of the aortic intima covered by plaques did not show significant differences between control and lacidipine-treated animals. These results suggest a direct antiatherosclerotic effect of lacidipine on proliferative lesions.

HMG CoA reductase inhibitors. In vivo effects on carotid intimal thickening in normocholesterolemic rabbits.

The in vivo activity of different 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (vastatins) on neointimal formation induced by insertion of a flexible collar around one carotid artery of normocholesterolemic rabbits was investigated. The contralateral carotid artery served as a sham control. Pravastatin, lovastatin, simvastatin, and fluvastatin were given mixed with food at daily doses of 20 mg/kg body wt for 2 weeks starting on the day of collar placement. The treatment with vastatins did not modify rabbit plasma cholesterol concentrations. The neointimal formation was assessed by measuring the cross-sectional thickness of intimal and medial tissues of fixed arteries with light microscopy. Fourteen days after collar placement, intimal hyperplasia (mostly cellular) was pronounced in treated carotid arteries. The intimal/medial (I/M) tissue ratio was 12-fold higher in treated arteries than in arteries without the collar (0.36 +/- 0.04 versus 0.03 +/- 0.02). Animals treated with lovastatin (n = 12), simvastatin (n = 12), and fluvastatin (n = 12) showed significantly less neointimal formation; I/M tissue ratios were 0.24 +/- 0.03, 0.20 +/- 0.03, and 0.17 +/- 0.03, respectively. The inhibition elicited by pravastatin (n = 12, 0.32 +/- 0.03) did not reach statistical significance. alpha-Actin antibody immunofluorescence analysis of serial sections revealed that cells present in the hyperplastic intima were mostly myocytes. Rates of intimal myocyte proliferation were also measured by incorporation of 5-bromo-2'-deoxyuridine, a thymidine analogue, into replicating DNA. Immunofluorescence analysis showed that 5-bromo-2'-deoxyuridine was actively incorporated into intimal myocytes after ++reinsertion of the collar, with a labeling index (percent of labeled myocytes) of 2.15 after 14 days.(ABSTRACT TRUNCATED AT 250 WORDS)