M.J. Gimeno

Fibroblasts from the transversalis fascia of young patients with direct inguinal hernias show constitutive MMP-2 overexpression.

ObjectiveTo determine the expression pattern of certain metalloproteinases (MMPs) known to be involved in the degradation of the extracellular matrix in cultured fibroblasts from the transversalis fascia (TF) of patients with inguinal hernia. Summary Background DataInguinal hernia is a common pathology, the cause of which remains unknown. It is, however, clear that the TF is one of the anatomical structures that may impede the formation of hernias, and particularly the direct type of hernia. In previous studies the authors found enhanced MMP-2 expression in TF specimens in vivo. The persistence of increased expression in cultured fibroblasts might support the idea of a genetic defect as the cause for this pathology. MethodsFibroblasts from the TF of patients with direct and indirect inguinal hernia were cultured and compared with those obtained from control TF in terms of MMP (MMP-2 and MMP-9) expression. ResultsSignificant active MMP-2 expression was shown by TF fibroblasts from young patients with direct hernias. These findings were confirmed by immunosorbent assay, immunoblotting, and zymography of the fibroblast culture media. No MMP-9 expression was detected. ConclusionThese results indicate that MMP-2 may be involved in the TF matrix degradative process in patients with direct hernia. The persistence of changes in MMP-2 levels in the cell cultures appears to suggest a genetic defect or irreversible change as the origin of this pathology rather than environmental factors, which may later participate in the development of the hernial process.

Expression of elastic components in healthy and varicose veins.

This study evaluates possible changes in the synthesis/degradation of elastic components of the vein wall in an attempt to explain the development of varicosis. Healthy and varicose saphenous veins were subjected to immunohistochemical analysis using anti-elastin, anti-fibrillin-1, anti-elastase, anti-transforming growth factor (TGF)-β and anti-latent TGFβ binding protein (LTBP)-2 monoclonal antibodies. In situ hybridization was performed using specific probes for tropoelastin and fibrillin-1. In healthy veins, elastin and fibrillin-1 showed even, overlapping distribution patterns indicating their particular abundance in the adventitia and at the intima/media interface. The expression of tropoelastin and fibrillin-1 was high in smooth muscle cells bordering the elastic laminae. Elastin, fibrillin-1, and cells expressing fibrillin-1 and tropoelastin mRNA showed a patchy disorganized pattern, particularly in the proximal varicose segments of patients under 50 years of age. Enhanced elastase activity was noted in both control and varicose specimens from elderly subjects. Varicose veins specimens showed greater LTBP-2 and TGF expression. Both molecules were detected in the subendothelium and the media, particularly in areas of marked injury. Our findings suggest that the development of the varicose condition involves a restructuring of the elastic component of the vein wall, perhaps as a consequence of changes in the transcription mechanisms of muscle layer cells.

Modulation of PECAM-1 (CD31) Expression in Human Endothelial Cells: Effect of IFNγ and IL-10

Intercellular contacts formed between endothelial cells (EC) permit the formation of a confluent monolayer and play a major role in the recruitment and the migration of leukocytes in the inflammatory response. It is currently accepted that cytokines are responsible for the signals involved in the induction of such endothelial alterations. The platelet EC adhesion molecule (PECAM-1), a specific component of EC junctions, is one of many molecules which participate in the regulation of EC interaction with blood components. Given that the regulatory mechanisms which affect the expression of this adhesion molecule are only partially understood, the aim of the present study was to compare the effects of two antagonistic inflammatory cytokines, interferon (IFN)-γ and interleukin (IL)-10, on the expression of PECAM-1. Human umbilical vein EC grown to subconfluence were stimulated with IL-10 (10 ng/ml) and/or IFNγ (100 U/ml) for 24 h. PECAM-1 expression was determined by FACScan and immunofluorescence. Morphological analysis of the cell cultures was performed by optical and scanning electron microscopy. In the presence of IL-10, no changes in cell growth and morphology or in the intensity of PECAM-1 expression were observed. However, when the cultures were treated with IFNγ, the EC acquired a fibroblast-like appearance, growth was disorganized and PECAM-1 disappeared from cell junctions. The mean intensity expression and the percentage of EC expression of this antigen were analyzed by flow cytometry and significantly decreased after culture in the presence of IFNγ. The simultaneous addition of IFNγ and IL-10 to the EC cultures induced modifications similar to those observed in the presence of IFNγ alone. Regulation of the expression of PECAM-1 with the subsequent functional implications seems to be dependent upon the IFNγ signal and it is unaffected by IL-10. The different effects shown by IL-10 and IFNγ on the expression of PECAM-1 in EC could reflect opposite regulatory actions of the inflammatory response induced by these cytokines.

El proceso de descongelación lenta mantiene la viabilidad de la pared arterial criopreservada

Resumen Investigaciones recientes han centrado su interes en los procesos de descongelacion como posibles inductores de los danos que hacen fracasar los injertos realizados con arterias criopreservadas. El objetivo del presente trabajo es, conocer el efecto de la descongelacion sobre la pared de vasos criopreservados a -80 oC. Arterias iliacas de cerdo (Mini Pig) fueron criopreservadas en un congelador biologico a -80 oC, en Medio Minimo Esencial con el 10% de dimetilsulfoxido, disminuyendo la temperatura 1 oC/min. Los vasos fueron almacenados 30 dias a -80 oC y una vez transcurrido este periodo de tiempo se sometieron a dos protocolos diferentes de descongelacion: descongelacion rapida, 5 minutos en un bano a 37 oC o descongelacion lenta, programada y automatizada, 2 horas; con un incremento de temperatura de 1 oC/m. hasta alcanzar la temperatura ambiente. Arterias frescas fueron utilizadas como controles. Se realizaron estudios morfologicos; microscopia optica y microscopia electronica de transmision y barrido, de los diferentes grupos y se valoro el dano celular mediante la tecnica TUNEL. En los vasos sometidos tanto a descongelacion lenta como a descongelacion rapida, existian zonas donde el endotelio estaba bien conservado, alternando con zonas totalmente denudadas. En la capa media, el grupo de descongelacion rapida presentaba una mayor desorganizacion celular, con presencia de zonas edematosas distribuidas por todo el espesor de la capa. La microscopia electronica de barrido mostraba una superficie luminal cubierta de celulas endoteliales globulares y pequenas areas denudadas que dejaban al descubierto una densa matriz subendotelial. Tras el proceso de criopreservaciou, el numero de celulas viables disminuye, produciendose un incremento de ceulas TUNEL-positivas. La viabilidad celular total resulto ser menor en aquellas arterias que habian sido sometidas al protocolo de descongelacion rapida. Por todo ello, podemos concluir que la descongelacion rapida, en comparacion con la descongelacion lenta, provoca danos mas acusados en la capa media de las arterias criopreservadas a -80 oC y disminuye la viabilidad celular de las mismas.

Inhibitor of angiotensin-converting enzyme modifies myointimal origin in an arterial autograft model

Pharmacologic modulation by an inhibitor of angiotensin-converting enzyme (IACE: cilazapril) of vascular proliferative response to a full-thickness arterial injury (autograft) was studied in rats. An arterial autograft 5 mm long was made in the right common iliac artery of 50 female Sprague-Dawley rats (weight 250-300 g) by microsurgical techniques. The animals were divided into two study groups: group I (controls), 20 animals that underwent arterial autograft but received no other treatment; and group II (cilazapril-treated), 20 rats that underwent arterial autograft and received cilazapril (Roche), 10 mg/day orally (p.o.) in an excipient of 2% arabic gum, for 4 days before operation. Animals were killed on postoperative days 7, 14, 21, 30, and 50, and grafts were studied by light microscopy, scanning and transmission electron microscopy, and morphometry. In the control group, the hyperplasic response had begun by postoperative day 14 and was established by postoperative day 50. In the medial layer, the muscle cells changed in phenotype from contractile to secretory cells. The adventitia had a highly proliferative appearance. In the cilazapril-treated group, fibrin deposits and platelets formed a layer on the internal elastic lamina. This layer appeared to evolve toward an intimal hyperplasia that became quantifiable by postoperative day 21. The medial layer was clearly thinned and showed intense accumulation of lipid microvacuoles, elastic degeneration, and vacuolized cells. Our results suggest that the use of an inhibitor of ACE modified the origin of the intimal hyperplasia in the arterial autograft model. Enhancement of the thrombogenicity of the luminal surface favors myointimal development by thrombus reorganization.