Understanding and manipulating the biological response to vascular implants.

Wound healing is a very complex and dynamic process that involves both stimulation and inhibition of cells and bioactive substances, resulting in a stable scar. Although the histological aspects of this response are well understood, the regulation of wound healing on a molecular level has not been completely elucidated. An "appropriate" healing response is crucial after vascular graft implantation to permit a functioning patent conduit. If the biological response is unfavorable, graft failure ensues. Clearly, it has been shown that the patency for prosthetic vascular implants is less favorable than for autologous implants in certain anatomic positions. However, the factors that promote this failure have not been fully identified. This article reviews the normal biological response to vascular graft healing as we understand it and provides some alternatives to manipulate the cellular milieu in an attempt to promote a more favorable healing response to prosthetic implantation.

Juxtalumenal location of plaque necrosis and neoformation in symptomatic carotid stenosis.

PURPOSE: The structural features that underlie carotid plaque disruption and symptoms are largely unknown. We have previously shown that the chemical composition and structural complexity of critical carotid stenoses are related to plaque size regardless of symptoms. To further determine whether the spatial distribution of individual plaque components in relation to the lumen corresponds to symptomatic outcome, we evaluated 99 carotid endarterectomy plaques. METHODS: Indications for operation were symptomatic disease in 59 instances (including hemispheric transient ischemic attack in 29, stroke in 19, and amaurosis fugax in 11) and angiographic asymptomatic stenosis > 75% in 40. Plaques removed after remote symptoms beyond 6 months were excluded. Histologic sections from the most stenotic region of the plaque were examined using computer-assisted morphometric analysis. The percent area of plaque cross-section occupied by necrotic lipid core with or without associated plaque hematoma, by calcification, as well as the distance from the lumen or fibrous cap of each of these features, were determined. The presence of foam cells, macrophages, and inflammatory cell collections within, on, or just beneath the fibrous cap was taken as an additional indication of plaque neoformation. RESULTS: The mean percent angiographic stenosis was 82% +/- 11% and 79% +/- 13% for the asymptomatic and symptomatic groups, respectively (p > 0.05). The necrotic core was twice as close to the lumen in symptomatic plaques when compared with asymptomatic plaques (0.27 +/- 0.3 mm vs 0.5 +/- 0.5 mm; p < 0.01). The percent area of necrotic core or calcification was similar for both groups (22% vs 26% and 7% vs 6%, respectively). There was no significant relationship to symptom production of either the distance of calcification from the lumen or of the percent area occupied by the lipid necrotic core or calcification. The number of macrophages infiltrating the region of the fibrous cap was three times greater in the symptomatic plaques compared with the asymptomatic plaques (1114 +/- 1104 vs 385 +/- 622, respectively, p < 0.009). Regions of fibrous cap disruption or ulceration were more commonly observed in the symptomatic plaques than in the asymptomatic plaques (32% vs 20%). None of the demographic or clinical atherosclerosis risk factors distinguished between symptomatic and asymptomatic plaques. CONCLUSIONS: These findings indicate that proximity of plaque necrotic core to the lumen and cellular indicators of plaque neoformation or inflammatory reaction about the fibrous cap are associated with clinical ischemic events. The morphologic complexity of carotid stenoses does not appear to determine symptomatic outcome but rather the topography of individual plaque components in relation to the fibrous cap and the lumen. Imaging techniques that precisely resolve the position of the necrotic core and evidence of inflammatory reactions within carotid plaques should help identify high-risk stenoses before disruption and symptomatic carotid disease.

Corticospinal neurons exhibit a novel pattern of cytoskeletal gene expression after injury.

We examined changes in the expression of major cytoskeletal protein mRNAs in adult hamster corticospinal neurons after axotomy. While a number of studies had determined that peripheral neurons exhibit major alterations in cytoskeletal gene expression after axotomy, no previous studies had addressed the question of whether or not intrinsic mammalian CNS neurons, which do not have the ability to successfully regenerate axons after injury, alter their expression of tubulin and neurofilament genes after injury. In the present study we used in situ hybridization methods to examine this issue. 35S-labeled cDNA probes for the low molecular weight neurofilament protein (NF-L) mRNA and an alpha-tubulin mRNA species (M alpha 1) were used for in situ hybridizations of sections of the sensorimotor cortex obtained 2, 7, and 14 days after unilateral axotomy of the corticospinal tract in the caudal medulla. Film as well as emulsion autoradiography showed dramatic decreases in both alpha-tubulin and NF-L mRNA levels within axotomized neurons in layer Vb of the sensorimotor cortex. Tubulin mRNA levels were decreased as early as 2 days after injury whereas NF-L mRNA levels were not decreased until later times. Ribosomal RNA (rRNA) levels in axotomized corticospinal neurons were also examined using in situ hybridization with a 35S-labeled rDNA probe. These studies showed only a slight decrease in rRNA levels in corticospinal neurons at 14 days after axotomy. Immunoblotting experiments of total protein from corticospinal axons in the medulla were performed to assess whether the axonal composition immediately proximal to the injury site reflected changes in cell body gene expression. Both alpha-tubulin and NF-L levels were found to decrease in corticospinal axons by 28 days after injury. These findings, to our knowledge, are the first to demonstrate that a class of mammalian CNS neurons have an intrinsically different cytoskeletal response to axonal injury than do PNS neurons. The failure to upregulate tubulin gene expression following injury may contribute to the ineffective regenerative response of these long-tract CNS neurons.

Vimentin mRNA expression increases after corticospinal axotomy in the adult hamster.

We examined changes in vimentin gene expression during Wallerian degeneration after corticospinal axotomy in the adult hamster. Vimentin, which is the product of a type III intermediate filament (IF) gene, is expressed in various cells of mesenchymal origin, including microvascular endothelial cells, microglia and developing astrocytes. While increases in vimentin protein have been observed after various types of central nervous system (CNS) injury, it is not known whether this increase is due to increased vimentin mRNA expression. There is also conflicting evidence as to which cells are expressing increased levels of vimentin. In the present study we used in situ hybridization and double-label immunofluorescence techniques to address these issues. A 35S-labeled vimentin cDNA probe was used for in situ hybridizations of brain stem sections obtained 2, 7 and 14 days after unilateral transection of the corticospinal tract in the caudal medulla of adult hamsters. Autoradiography showed that an increase in vimentin mRNA associated with the degenerating corticospinal tract occurred by 2 days after axotomy and that the levels remained elevated for at least 14 days. Immunoblotting and immunocytochemical studies indicated that vimentin protein levels were increased in the degenerating corticospinal tract. Double-label immunofluorescence revealed many vimentin-positive cells and processes that were also labeled with GFAP antibody. In addition, cells and processes that were vimentin-negative but GFAP-positive were also found in the degenerating tract. We suggest that the reactive cells which possessed both vimentin and GFAP were reactive astrocytes of astroblastic origin while those that expressed only GFAP were derived from mature astrocytes. Other vimentin-positive cells/processes did not label with anti-GFAP and thus were either microglial, endothelial or inflammatory cells. These results demonstrate that an increase in vimentin mRNA occurs during Wallerian degeneration after corticospinal axotomy and that this increase is likely to be due to contributions from more than one cell type.

Changes in glial fibrillary acidic protein mRNA expression after corticospinal axotomy in the adult hamster.

We examined changes in the expression of glial fibrillary acidic protein (GFAP) mRNA during Wallerian degeneration in the corticospinal system of the adult Golden hamster following axotomy. GFAP is the product of a type III intermediate filament (IF) gene that is expressed specifically in mature astrocytes. A well-studied component of a complex response termed reactive astrogliosis that occurs after various types of CNS injury is the increased production of astrocytic processes filled with GFAP-containing IFs. While increased expression of GFAP during reactive astrogliosis has been well established at the protein level, little is known about whether or not changes in GFAP mRNA levels occur after CNS injury. In the present study we used in situ hybridization methods to examine this issue. A 35S-labeled mouse GFAP cDNA probe was used for in situ hybridizations of sections of the brain stem obtained 2, 7, and 14 days after unilateral transections of the corticospinal tract in the caudal medulla. Film as well as emulsion autoradiography showed a dramatic increase in GFAP mRNA labeling associated with the degenerating corticospinal tract. GFAP mRNA levels were already dramatically increased in the injured corticospinal tract by 2 days post axotomy and remained elevated at 14 days. Interestingly, in addition to the robust increase in GFAP mRNA levels specifically associated with the degenerating tract, a diffuse increase in GFAP mRNA labeling was observed throughout the grey matter of the brain stem at 2 days post-axotomy, but not after this time. Immunoblotting and immunocytochemical experiments verified that the increased GFAP mRNA levels in the degenerating corticospinal system were accompanied by an increased expression of the protein. These results demonstrate that an increase in GFAP mRNA levels occurs during Wallerian degeneration in the CNS and suggest that increased expression of the GFAP gene is a major contributor to CNS scarring that results after direct traumatic injury.