Alexander Black

Pravastatin Reduces Marfan Aortic Dilation

Background— The sequelae of aortic root dilation are the lethal consequences of Marfan syndrome. The root dilation is attributable to an imbalance between deposition of matrix elements and metalloproteinases in the aortic medial layer as a result of excessive transforming growth factor-beta signaling. This study examined the efficacy and mechanism of statins in attenuating aortic root dilation in Marfan syndrome and compared effects to the other main proposed preventative agent, losartan. Methods and Results— Marfan mice heterozygous for a mutant allele encoding a cysteine substitution in fibrillin-1 (C1039G) were treated daily from 6 weeks old with pravastatin 0.5g/L or losartan 0.6 g/L. The end points of aortic root diameter (n=25), aortic thickness, and architecture (n=10), elastin volume (n=5), dp/dtmax (maximal rate of change of pressure) (cardiac catheter; n=20), and ultrastructural analysis with stereology (electron microscopy; n=5) were examined. The aortic root diameters of untreated Marfan mice were significantly increased in comparison to normal mice (0.161±0.001 cm vs 0.252±0.004 cm; P<0.01). Pravastatin (0.22±0.003 cm; P<0.01) and losartan (0.221±0.004 cm; P<0.01) produced a significant reduction in aortic root dilation. Both drugs also preserved elastin volume within the medial layer (pravastatin 0.23±0.02 and losartan 0.29±0.03 vs untreated Marfan 0.19±0.02; P=0.01; normal mice 0.27±0.02). Ultrastructural analysis showed a reduction of rough endoplasmic reticulum in smooth muscle cells with pravastatin (0.022±0.004) and losartan (0.013±0.001) compared to untreated Marfan mice (0.035±0.004; P<0.01). Conclusions— Statins are similar to losartan in attenuating aortic root dilation in a mouse model of Marfan syndrome. They appear to act through reducing the excessive protein manufacture by vascular smooth muscle cells, which occurs in the Marfan aorta. As a drug that is relatively well-tolerated for long-term use, it may be useful clinically.

Distribution of myofibroblasts, smooth muscle-like cells, macrophages, and mast cells in mitral valve leaflets of dogs with myxomatous mitral valve disease.

OBJECTIVE To map the cellular distribution and phenotypic alteration of the predominant stromal cell population throughout the entire valve length of dogs with myxomatous mitral valve disease (MMVD). SAMPLE POPULATION 31 mitral valve complexes (ie, mitral valve leaflets) collected from 4 clinically normal dogs and 27 dogs with MMVD of varying severity. PROCEDURES A combination of standard histologic and immunohistochemical techniques was used to identify pathologic changes, the presence of mast cells, and the density and distribution of cells expressing vimentin, desmin, alpha-smooth muscle actin (alpha-SMA), smooth muscle myosin, and the macrophage marker MAC387. RESULTS Vimentin-positive cells predominated in the mitral valve leaflets from clinically normal dogs and were located throughout the leaflet, but cell density was appreciably decreased with disease progression, and minimal cell numbers were found in distinct myxomatous areas. Cells that were positive for alpha-SMA were uncommon in the mitral valve leaflets from clinically normal dogs and only seen in appreciable numbers in mitral valves of dogs with severe late-stage disease, in which cells were typically located close to the ventricularis valve surface. A slight increase in mast cell numbers was observed in the distal zone of affected leaflets. CONCLUSIONS AND CLINICAL RELEVANCE Activated-myofibroblasts (alpha-SMA-positive cells) were increased and inactive-myofibroblasts (vimentin-positive cells) were reduced in mitral valve leaflets of dogs with MMVD, compared with that of clinically normal dogs. Impact on Human Medicine-This is the first description of spatial and temporal alterations in mitral valve cells of any species with MMVD and has clinical importance in the understanding of disease development in dogs and humans.

Reference Models for Mitral Valve Tissue Engineering Based on Valve Cell Phenotype and Extracellular Matrix Analysis

The advance of mitral valve repair techniques through tissue engineering is impeded by the lack of information regarding the cellular and extracellular components of the mitral valve. The present study aims to expand our understanding of the mitral valve structure by analysing the synthesis of extracellular matrix (ECM) proteins and the expression of nitric oxide synthase (NOS). Valvular endothelial cells (VECs) and valvular interstitial cells (VICs) were isolated from porcine mitral valves. Immunochemical staining of ECM components, including type I, II, III, IV and V collagen, laminin, fibronectin, elastin and chondroitin sulphate (CS), was performed on both mitral valve tissue and cell cultures. Reverse transcription polymerase chain reaction and immunochemistry were used to analyse NOS expression in native valve and in culture. Both VECs and VICs synthesised the basement membrane components, laminin and type IV collagen both in vivo and in vitro, amongst other fibrous ECM proteins. Synthesis of type I collagen and CS was absent in VEC cultures. Each cell type had a characteristic profile of NOS expression. VECs synthesised endothelial NOS both in vivo and in vitro, with a minority of VICs expressing neuronal NOS in vitro. The present study reports newly recognised aspects of the mitral valve structure and the in vitro behaviour of mitral valve cell populations based on ECM synthesis and NOS expression. The presented profiles can be used as base tools for the generation of data necessary for the selection of ideal cell sources and for the design of appropriate scaffolds for the development of effective tissue-engineered mitral valves.

Morphological changes to endothelial and interstitial cells and to the extra-cellular matrix in canine myxomatous mitral valve disease (endocardiosis)

Morphological and functional changes in endothelial and interstitial cells are considered central to myxomatous degeneration of the canine mitral valve (endocardiosis). The aim of this study was to describe and quantify changes in valve endothelial cells (VECs), interstitial cells (VICs) and the extra-cellular matrix (ECM) of the sub-endothelial zone of diseased valves using a combination of transmission electron microscopy, stereology and computer-aided image analysis. Marked degradation of the endothelium was evident in diseased valves, which coincided with significant degradation of the local ECM (P<0.001). There were decreases and increases in the numbers of VECs and VICs, respectively, in diseased valves, with particular accumulation of VICs subjacent to the valve surface (P<0.01). Overall, VICs were more pleomorphic than VECs in both normal and diseased valves, but for VECs, the degree of pleomorphism was significantly different in diseased valves (P<0.0001). The findings of the study confirm that canine myxomatous mitral valve disease is associated with marked endothelial damage, with attendant proliferation of subjacent activated myofibroblasts. The fact that similar endothelial changes are present in normal valves suggests these processes not only contribute to valve pathology, but may also represent life-long valve remodelling.

Variation and specialisation of the forcipular apparatus of centipedes (Arthropoda: Chilopoda): a comparative morphometric and microscopic investigation of an evolutionary novelty.

The forcipules of centipedes are the only known example in the animal kingdom of an evolutionary transition from walking legs to venom-injecting appendages. They provide a classic case of an evolutionary novelty under most (but not all) definitions of that concept. Although there is a reasonable literature on forcipules, and on the forcipular segment more generally, it is fragmentary and scattered. Also, many previous studies have been based on a single species and hence have no comparative component. Here, we build on this earlier literature by providing detailed qualitative and quantitative information on the forcipular segments of representatives of the five extant orders of centipedes. Our results reveal notable differences between the orders - as well as considerable variation within some of them. The pattern of inter-group differences can be used to infer, albeit cautiously, a major evolutionary trend from a presumed scutigeromorph-like last common ancestor (LCA), in which the forcipules were probably leg-like (as in present-day scutigeromorphs) to a more specialized claw-like structure with movement restricted to the horizontal plane. This morphological trend may reflect an ecological trend from open-habitat ambush predation to leaf-litter and subterranean predatory opportunism.

Appraisal and Consequences of Cadaver Dissection

Abstract First exposure to human cadaver dissection has the potential to be an actual stressor which can cause psychological trauma. This study examines the relationship between anatomy students’ experience of this potential stressor and various psychological and personal factors. Questionnaires measuring emotional reactions to cadaver dissection, coping strategies, personality and attitudes to death were administered to anatomy students at two medical schools immediately after their first exposure to human cadaver dissection. Emotional reactions to recalling this experience were assessed 4 months later. Data on these variables were obtained from 141 students. Students found the experience mostly challenging and, on average, did not report serious emotional difficulties. However, a minority of students (10/141) experienced serious adverse consequences. It is possible that the typical student who undertakes an anatomy course is already psychologically prepared for such transactions. However, low cost desensitization programs could be made available for the minority of individuals who may experience adverse reactions in this situation.

Development of the venom ducts in the centipede Scolopendra: an example of recapitulation

In contrast to previous claims that (a) there is a law of recapitulation and, conversely, (b) recapitulation never happens, the evolutionary repatterning of development can take many forms, of which recapitulation is one. Here, we add another example to the list of case studies of recapitulation. This example involves the development of the venom claws (forcipules) in the centipede Scolopendra subspinipes mutilans, and in particular the development of the duct through which venom flows from the gland that produces it (proximal) to the opening called the meatus (distal) through which it is injected into prey. Most of the information we present is from early postembryonic stages—these have been neglected in previous work on centipede development. We show that the venom ducts arise from sutures that are invaginations of the cuticle. In S. s. mutilans, the invagination in each forcipule forms into a tubular structure that detaches itself from the exoskeleton and moves toward the center of the forcipule. This is in contrast to extant Scutigera, and also, probably, Scolopendras extinct Scutigera‐like ancestors, where the duct remains attached to the cuticle of throughout development. Thus, S. s. mutilans exhibits a recapitulatory repatterning of development.

Short-, mid-, and long-term effects of a polymer-free tacrolimus-eluting stent in a porcine coronary model

Stent-based delivery of tacrolimus has shown neointimal hyperplasia and restenosis reduction; FK506 is a water insoluble macrolide immunosuppressant. The purpose of this study was to evaluate acute and chronic tissue response to a polymer-free FK506 drug-eluting stent implantation in a porcine coronary artery model. Seventy-eight nonatherosclerotic minipigs underwent successful placement of 134 stents (control n = 56; FK506 (1.5 microg/mm(2)) n = 44; FK506 (2.6 microg/mm(2)) n = 34) at 7, 15, 30, 90, or 180 days. Endothelialisation was almost complete at 7 days, complete at 15 days. At 30 and 90 days, mean neointimal thickness, neointimal area, and % stenosis was significantly less for drug-eluting stents compared with controls. At 180 days, histomorphometric values were similar for eluting and control stents. The FK506-eluting stent allows for a complete re-endothelialisation at 15 days and favorably moderate neointimal hyperplasia at 30 and 90 days in the porcine coronary model. Because of a possible limited bioavailability of FK506, long-term inhibition of neointimal formation was not sustained at the considered follow-up.

Evaluation of innervation of the mitral valves and the effects of myxomatous degeneration in dogs

OBJECTIVE To map aspects of the innervation of the mitral valve complex and determine any association with the development or progression of myxomatous mitral valve disease (MMVD) in dogs. SAMPLE POPULATION Septal mitral valve leaflets from 11 dogs aged 6 months to > 10 years. PROCEDURES Expression of protein gene product 9.5 (general neuronal marker), tyrosine hydroxylase (adrenergic innervation marker), vasoactive intestinal peptide (parasympathetic innervation marker), and calcitonin gene-related peptide (sensory innervation marker) was assessed by use of a standard immunohistochemical technique. Innervation was assessed qualitatively and semiquantitatively. Differences between valvular zones and between groups were analyzed statistically. RESULTS MMVD was present in leaflets of all dogs > or = 5 years of age. Innervation was confirmed in all leaflets but was markedly reduced in leaflets of dogs > 10 years of age. Innervation was most dense at the base of valves and mainly associated with the epimysial, perimysial, and endomysial layers of the muscle and blood vessels within the valve. Innervation was reduced within the middle zone of the valve and lacking at the free edge. Innervation was not identified at the tip of the leaflet, the free edge, or the chordae. Nerve fibers were mostly sympathetic, with the remainder being parasympathetic or sensory. Existence of MMVD did not alter the pattern or density of innervation. CONCLUSIONS AND CLINICAL RELEVANCE Mitral valve leaflets in the study dogs were innervated, with most of the nerve fibers associated with the myocardium in the valve base. Development of MMVD appeared to precede the reduction of innervation associated with advancing age.

Cell maceration scanning electron microscopy and computer-derived porosity measurements in assessment of connective tissue microstructure changes in the canine myxomatous mitral valve

Canine myxomatous mitral valve disease is associated with changes in the valve extracellular matrix (ECM). The aim of this study was to examine the use of cell macerated scanning electron microscopy (CMSEM) in evaluating ECM changes in a small sample of valves and to quantify these changes using computer-aided image analysis of sample porosity (a measure of structural disorganisation and collagen loss). The distinct layered structure of the de-cellularised matrix could be seen in the normal valve and there were marked changes in layers and ECM organisation as the disease progressed. Clearly visible and quantifiable, statistically significant changes were found in valve porosity across the entire leaflet thickness and particularly in the valve mid and distal zones. All of these changes are presumed to affect the mechanical function of the valve. In conclusion, CMSEM with computed image analysis can be used to visualise and measure tissue structural changes in a quasi-3-dimensional manner in normal and diseased tissues.