Mitochondrial DNA deletions and depletion within paraspinal muscles.

AIMS: Although mitochondrial abnormalities have been reported within paraspinal muscles in patients with axial weakness and neuromuscular disease as well as with ageing, the basis of respiratory deficiency in paraspinal muscles is not known. This study aimed to determine the extent and basis of respiratory deficiency in paraspinal muscles from cases undergoing surgery for degenerative spinal disease and post mortem cases without a history of spinal disease, where age-related histopathological changes were previously reported. METHODS: Cervical and lumbar paraspinal muscles were obtained peri-operatively from 13 patients and from six post mortem control cases (age range 18-82 years) without a neurological disease. Sequential COX/SDH (mitochondrial respiratory chain complex IV/complex II) histochemistry was performed to identify respiratory-deficient muscle fibres (lacking complex IV with intact complex II activity). Real-time polymerase chain reaction, long-range polymerase chain reaction and sequencing were used to identify and characterize mitochondrial DNA (mtDNA) deletions and determine mtDNA copy number status. Mitochondrial respiratory chain complex subunits were detected by immunohistochemistry. RESULTS: The density of respiratory-deficient fibres increased with age. On average, 3.96% of fibres in paraspinal muscles were respiratory-deficient (range 0-10.26). Respiratory deficiency in 36.8% of paraspinal muscle fibres was due to clonally expanded mtDNA deletions. MtDNA depletion accounted for further 13.5% of respiratory deficiency. The profile of immunohistochemically detected subunits of complexes was similar in respiratory-deficient fibres with and without mtDNA deletions or mtDNA depletion. CONCLUSIONS: Paraspinal muscles appeared to be particularly susceptible to age-related mitochondrial respiratory chain defects. Clonally expanded mtDNA deletions and focal mtDNA depletion may contribute towards the development of age-related postural abnormalities.

Identification and investigation of mitochondria lacking cytochrome c oxidase activity in axons.

Mitochondrial defects have been implicated in the degeneration of axons in a number of CNS disorders, including multiple sclerosis. Uniquely, mitochondria harbor the only non-nuclear DNA (mitochondrial DNA or mtDNA), which encodes functionally important subunits of the respiratory chain. The pattern of mitochondrial respiratory chain subunit expression provides important clues to the underlying mechanism of mitochondrial injury. In snap frozen tissue mitochondrial respiratory chain complex IV or cytochrome c oxidase (COX) activity may be determined using a well-established histochemical technique, COX histochemistry. Lack of COX activity may be the result of mtDNA mutations, degradation of transcripts of subunits, modification of subunits or inhibition of complexes. Mitochondria lacking complex IV activity, however, have not been further explored within axons in CNS disorders. By combining COX histochemistry with immunofluorescent labeling of mitochondrial proteins we describe a method to identify mitochondria lacking complex IV activity in CNS tissue and locate inactive mitochondria to axons using confocal microscopy. Inactive axonal mitochondria may then be further investigated using confocal microscopy to define the pattern of mitochondrial respiratory chain complex subunit expression. Our technique may be used to gain important clues to the underlying mechanisms of mitochondrial injury within axons in a number of CNS disorders and relevant animal models.

Gene expression profile of the fibrotic response in the peritoneal cavity.

The cellular response to materials implanted in the peritoneal cavity has been utilised to produce tissue for grafting to hollow smooth muscle organs (blood vessels, bladder, uterus and vas deferens). To gain insight into the regulatory mechanisms involved in encapsulation of a foreign object, and subsequent differentiation of encapsulating cells, the present study used microarray technology and real-time RT-PCR to identify the temporal changes in gene expression associated with tissue development. Immunohistochemical analysis showed that 3-7 days post-implantation of foreign objects (cubes of boiled egg white) into rats, they were encapsulated by tissue comprised primarily of haemopoietic (CD45(+)) cells, mainly macrophages (CD68(+), CCR1(+)). By day 14, tissue capsule cells no longer expressed CD68, but were positive for myofibroblast markers alpha-smooth muscle (SM) actin and SM22. In accordance with these results, gene expression data showed that early capsule (days 3-7) development was dominated by the expression of monocyte/macrophage-specific genes (CD14, CSF-1, CSF-1R, MCP-1) and pro-inflammatory mediators such as transforming growth factor (TGF-beta). As tissue capsule development progressed (days 14-21), myofibroblast-associated and pro-fibrotic genes (associated with TGF-beta and Wnt/beta-catenin signalling pathways, including Wnt 4, TGFbetaRII, connective tissue growth factor (CTGF), SMADs-1, -2, -4 and collagen-1 subunits) were significantly up-regulated. The up-regulation of genes associated with Cardiovascular and Skeletal and Muscular System Development at later time-points suggests the capacity of cells within the tissue capsule for further differentiation to smooth muscle, and possibly other cell types. The identification of key regulatory pathways and molecules associated with the fibrotic response to implanted materials has important applications not only for optimising tissue engineering strategies, but also to control deleterious fibrotic responses.

Tat-specific binding IgG and disease progression in HIV type 1-infected Ugandans.

There are data to suggest that both the humoral and cellular immune responses directed against Tat are beneficial in delaying HIV disease progression. We examined the association between the occurrence of Tat-specific binding antibodies (Abs) and different parameters of HIV-1 disease progression. We generated eight Tat proteins, derived from HIV-1 subtypes A, B, C, and D, and circulating recombinant form CRF01_AE. These proteins were used to screen for Tat-specific binding Abs by an ELISA. Using five Tat proteins, we investigated whether the occurrence of Tat-specific Abs within 2 years after seroconversion for the majority, affected disease progression over time among 126 participants using survival analysis and rate of CD4 decline. Of these, 52 participants with a sample at 1.5 and 4.5 years after seroconversion were further examined to study the effect of Tat-specific Ab loss or maintenance on disease progression. Finally, using all the eight Tat proteins, we also investigated whether specific Abs to these Tat proteins among 48 participants, grouped as rapid progressors (RP, n = 26) and long-term survivors (LTS, n = 22) according to their CD4 decline over time, affected disease progression. Survival analysis did not reveal any evidence of protection from progression by Tat-specific Abs. Comparison of rate of CD4 declines between individuals with and without Abs to any Tat protein showed only a small and borderline significant advantage of having Tat-specific Abs (p = 0.043). There was no correlation between either loss or maintenance of Tat-specific Abs and disease progression. Comparison of LTS with RP showed no evidence that Tat-specific Abs slows participants' disease progression. This study showed no evidence of a protective effect of having Tat-specific Abs among these Ugandan subjects.

Development of tissue engineered vascular grafts.

Vascular bypass grafting is a commonly performed procedure for ischemic heart disease and peripheral vascular disease. However, approximately one in fourteen patients do not have suitable autologous arteries or veins available for grafting. Synthetic vascular grafts were introduced in the 1960s to overcome these problems, but while they perform adequately in high-flow, large-diameter vessel settings they are generally not suited to low-flow, small-diameter vessels. Tissue engineering is a relatively new discipline that offers the potential to create replacement structures from autologous cells and biodegradable polymer scaffolds. Because tissue engineering constructs contain living cells, they may have the potential to grow, self-repair, and self-remodel. Therefore, recently there has been much interest in the use of this technique to produce low-flow small-diameter arteries. The latest and most exciting developments in this area involve the use of multipotent stem cells as a cell source for tissue engineering of vascular grafts (both in vivo and in vitro).

Bovine spongiform encephalopathy.

Early epidemiological studies identified bovine spongiform encephalopathy as a feed-borne infection associated with infected meat-and-bone meal in animal feed. The infection may have derived from scrapie in sheep, a spontaneous genetic mutation in cattle, or a transmissible spongiform encephalopathy in another mammalian species. Experimental work on the risk of transmission has necessarily tried to identify risk materials and their infectivity levels, the nature and size of species barriers, the infectious dose, the route of infection, the strain of the agent and the genotype of the animals at risk. The identification of levels of infection in cattle tissues has aided the removal of risk materials from the human and animal food chains. Maternally associated transmission is unlikely to maintain an outbreak, but the offspring of clinical cases appear to be at greater risk when the rate of food-borne exposure is high. Studies of embryo transfer have not shown infection to be transmitted by this means. While the control measures appeared to be straightforward, compliance has at times proved difficult to enforce and quantify. This has necessitated more extensive prohibitions, aggressive enforcement and thorough auditing of compliance levels.

Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteins.

Smooth muscle cells (SMC) exhibit a functional plasticity, modulating from the mature phenotype in which the primary function is contraction, to a less differentiated state with increased capacities for motility, protein synthesis, and proliferation. The present study determined, using Western analysis, double-label immunofluorescence and confocal microscopy, whether changes in phenotypic expression of rabbit aortic SMC in culture could be correlated with alterations in expression and distribution of structural proteins. "Contractile" state SMC (days 1 and 3 of primary culture) showed distinct sorting of proteins into subcellular domains, consistent with the theory that the SMC structural machinery is compartmentalised within the cell. Proteins specialised for contraction (alpha-SM actin, SM-MHC, and calponin) were highly expressed in these cells and concentrated in the upper central region of the cell. Vimentin was confined to the body of the cell, providing support for the contractile apparatus but not co-localising with it. In line with its role in cell attachment and motility, beta-NM actin was localised to the cell periphery and basal cortex. The dense body protein alpha-actinin was concentrated at the cell periphery, possibly stabilising both contractile and motile apparatus. Vinculin-containing focal adhesions were well developed, indicating the cells' strong adhesion to substrate. In "synthetic" state SMC (passages 2-3 of culture), there was decreased expression of contractile and adhesion (vinculin) proteins with a concomitant increase in cytoskeletal proteins (beta-non-muscle [NM] actin and vimentin). These quantitative changes in structural proteins were associated with dramatic changes in their distribution. The distinct compartmentalisation of structural proteins observed in "contractile" state SMC was no longer obvious, with proteins more evenly distributed throughout the cytoplasm to accommodate altered cell function. Thus, SMC phenotypic modulation involves not only quantitative changes in contractile and cytoskeletal proteins, but also reorganisation of these proteins. Since the cytoskeleton acts as a spatial regulator of intracellular signalling, reorganisation of the cytoskeleton may lead to realignment of signalling molecules, which, in turn, may mediate the changes in function associated with SMC phenotypic modulation.

Reorganization of structural proteins in vascular smooth muscle cells grown in collagen gel and basement membrane matrices (Matrigel): a comparison with their in situ counterparts.

When smooth muscle cells are enzyme-dispersed from tissues they lose their original filament architecture and extracellular matrix surrounds. They then reorganize their structural proteins to accommodate a 2-D growth environment when seeded onto culture dishes. The aim of the present study was to determine the expression and reorganization of the structural proteins in rabbit aortic smooth muscle cells seeded into 3-D collagen gel and Matrigel (a basement membrane matrix). It was shown that smooth muscle cells seeded in both gels gradually reorganize their structural proteins into an architecture similar to that of their in vivo counterparts. At the same time, a gradual decrease in levels of smooth muscle-specific contractile proteins (mainly smooth muscle myosin heavy chain-2) and an increase in beta-nonmuscle actin occur, independent of both cell growth and extracellular matrix components. Thus, smooth muscle cells in 3-D extracellular matrix culture and in vivo have a similar filament architecture in which the contractile proteins such as actin, myosin, and alpha-actinin are organized into longitudinally arranged "myofibrils" and the vimentin-containing intermediate filaments form a meshed cytoskeletal network. However, the myofibrils reorganized in vitro contain less smooth muscle-specific and more nonmuscle contractile proteins.

Circulating bone marrow cells can contribute to neointimal formation.

To examine the source of smooth muscle-like cells during vascular healing, C57BL/6 (Ly 5.2) female mice underwent whole body irradiation followed by transfusion with 10(6) nucleated bone marrow cells from congenic (Ly 5.1) male donors. Successful repopulation (88.4 +/- 4.9%) by donor marrow was demonstrated in the female mice by flow cytometry with FITC-conjugated A20.1/Ly 5.1 monoclonal antibody after 4 weeks. The arteries of the female mice were then subjected to two types of insult: (1) The iliac artery was scratch-injured by 5 passes of a probe causing severe medial damage. After 4 weeks, the arterial lumen was obliterated by a cell-rich neointima, with cells containing alpha smooth muscle actin present around the residual lumen. Approximately half of these cells were of male donor origin, as evidenced by in situ hybridization with a Y-chromosome-specific probe. (2) In an organized arterial thrombus formed by inserting an 8-0 silk suture into the left common carotid artery, donor cells staining with alpha smooth muscle actin were found in those arteries sustaining serious damage but not in arteries with minimal damage. Our results suggest that bone marrow-derived cells are recruited in vascular healing as a complementary source of smooth muscle-like cells when the media is severely damaged and few resident smooth muscle cells are available to effect repair.

Molecular basis by which garlic suppresses atherosclerosis.

The aim of this study was to determine the mechanism by which the aged garlic extract "Kyolic" has a protective effect against atherosclerosis. Plasma cholesterol of rabbits fed a 1% cholesterol-enriched diet for 6 wk was not reduced by supplementation with 800 microL Kyolic/(kg body. d). In spite of this, Kyolic reduced by 64% (P < 0.05) the surface area of the thoracic aorta covered by fatty streaks and significantly reduced aortic arch cholesterol. Kyolic also significantly inhibited by approximately 50% the development of thickened, lipid-filled lesions in preformed neointimas produced by Fogarty 2F balloon catheter injury of the right carotid artery in cholesterol-fed rabbits. In vitro studies found that Kyolic completely prevented vascular smooth muscle phenotypic change from the contractile, high volume fraction of filament (V(v)myo) state, and inhibited proliferation of smooth muscle cells in the synthetic state with a 50% effective dose (ED(50)) of 0.2%. Kyolic also slightly inhibited the accumulation of lipid in cultured macrophages but not smooth muscle, and had no effect on the expression of adhesion molecules on the surface of the endothelium or the adherence of leukocytes. It is concluded that Kyolic exerts antiatherogenic effects through inhibition of smooth muscle phenotypic change and proliferation, and by another (unclarified) effect on lipid accumulation in the artery wall.

Relationship of glycosaminoglycan and matrix changes to vascular smooth muscle cell phenotype modulation in rabbit arteries after acute injury.

PURPOSE: The phenotype of vascular smooth muscle cells (SMCs) is altered in several arterial pathologies, including the neointima formed after acute arterial injury. This study examined the time course of this phenotypic change in relation to changes in the amount and distribution of matrix glycosaminoglycans. METHODS: The immunochemical staining of heparan sulphates (HS) and chondroitin sulphates (CS) in the extracellular matrix of the arterial wall was examined at early points after balloon catheter injury of the rabbit carotid artery. SMC phenotype was assessed by means of ultrastructural morphometry of the cytoplasmic volume fraction of myofilaments. The proportions of cell and matrix components in the media were analyzed with similar morphometric techniques. RESULTS: HS and CS were shown in close association with SMCs of the uninjured arterial media as well as being more widespread within the matrix. Within 6 hours after arterial injury, there was loss of the regular pericellular distribution of both HS and CS, which was associated with a significant expansion in the extracellular space. This preceded the change in ultrastructural phenotype of the SMCs. The glycosaminoglycan loss was most exaggerated at 4 days, after which time the HS and CS reappeared around the medial SMCs. SMCs of the recovering media were able to rapidly replace their glycosaminoglycans, whereas SMCs of the developing neointima failed to produce HS as readily as they produced CS. CONCLUSIONS: These studies indicate that changes in glycosaminoglycans of the extracellular matrix precede changes in SMC phenotype after acute arterial injury. In the recovering arterial media, SMCs replace their matrix glycosaminoglycans rapidly, whereas the newly established neointima fails to produce similar amounts of heparan sulphates.

Neointimal formation by circulating bone marrow cells.

The origin of smooth muscle cells involved in vascular healing was examined. Eighteen C57BL/6 (Ly 5.2) female mice underwent whole body irradiation followed by transfusion with 106 bone nucleated marrow cells from congenic (Ly 5.1) male donors. Successful repopulation by donor marrow was demonstrated after 4 weeks by flow cytometry with FITC-conjugated A20.1/Ly 5.1 monoclonal antibody. The iliac artery of six of the chimeric mice was scratch-injured by five passes of a probe, causing severe medial damage. After 4 weeks the arterial lumen was obliterated by a cell-rich neointima, with alpha-smooth muscle actin-containing cells present around the residual lumen. Approximately half of these cells were of male donor origin, as evidenced by in situ hybridization with a Y chromosome-specific probe. An organized arterial thrombus was formed in the remaining 12 chimeric mice by inserting an 8-0 silk suture into the left common carotid artery. Donor cells staining with alpha-smooth muscle actin were found in those arteries sustaining serious damage but not in arteries with minimal damage. Our results suggest that bone marrow-derived cells are recruited in vascular healing as a complementary source of smooth muscle-like cells when the media is severely damaged and few resident smooth muscle cells are available to effect repair.

A role for rho in smooth muscle phenotypic regulation.

The role of the small GTP-binding protein Rho in the process of smooth muscle cell (SMC) phenotypic modulation was investigated using cultured rabbit aortic SMCs. Both Rho transcription and Rho protein expression were high for the first 3 days of culture ("contractile" state cells), with expression decreasing after change to the "synthetic" state and peaking upon return to the contractile phenotype. Activation of Rho (indicated by translocation to the membrane) also peaked upon return to the contractile state and was low in synthetic state SMCs. Transient transfection of synthetic state rabbit SMCs with constitutively active Rho (vall4rho) caused a dramatic decrease in cell size and reorganization of cytoskeletal proteins to resemble those of the contractile phenotype; alpha-actin and myosin adopted a tightly packed, highly organized arrangement, whereas vimentin localized to the immediate perinuclear region and focal adhesions were enlarged. Conversely, specific inhibition of endogenous Rho, by expression of C3 transferase, resulted in the complete loss of actin and myosin filaments without affecting the distribution of vimentin. Focal adhesions were reduced in number. Thus, Rho plays a key role in regulating SMC phenotypic expression.

Detection of Escherichia coli O157:H7 in soil and water using multiplex PCR.

AIMS: To evaluate the suitability of a multiplex PCR-based assay for sensitive and rapid detection of Escherichia coli O157:H7 in soil and water. METHODS AND RESULTS: Soil and water samples were spiked with E. coli O157:H7 and subjected to two stages of enrichment prior to multiplex PCR. Detection sensitivities were as high as 1 cfu ml(-1) drinking water and 2 cfu g(-1) soil. Starvation of E. coli O157:H7 for 35 d prior to addition to soil did not affect the ability of the assay to detect initial cell numbers as low as 10 cfu g(-1) soil. Use of an 8-h primary enrichment enabled detection of as few as 6 cfu g(-1) soil, and 10(4) cfu g(-1) soil with a 6-h primary enrichment. When soil was inoculated with 10(5) cfu g(-1), the PCR assay indicated persistence of E. coli O157:H7 during a 35 d incubation. However, when soil was inoculated with lower numbers of pathogen, PCR amplification signals indicated survival to be dependent on cell concentration. CONCLUSIONS: A multiplex PCR-based assay, in combination with an enrichment strategy enabled sensitive and rapid detection of E. coli O157:H7 in soil and water. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to sensitively detect E.coli O157:H7 in environmental material within one working day represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen.

Haemopoietic origin of myofibroblasts formed in the peritoneal cavity in response to a foreign body.

This study utilized both in vivo and in vitro techniques to investigate whether cells of bone marrow origin can differentiate into smooth muscle-like cells (myofibroblasts) with contractile filaments and proteins. Female C57BL/6 mice expressing the Ly5.2 antigen on the surface of their haemopoietic cells had four pieces of silastic tubing (3 x 0.5 mm outer diameter) or boiled blood clot (2-3 mm diameter) placed in their peritoneal cavity. After 3, 5, 7 and 14 days (n = 4/group) the implants were removed and those that had remained free-floating were processed for light microscopy, immunohistochemistry and electron microscopy. In the first 3-5 days, rounded cells adhered to the entire surface of the tubing then flattened. These cells stained with fluoresceinated antibodies to Ly5.2 showing that they were derived from haemopoietic cells. By 14 days the cells had become elongated and multilayered in a collagen matrix, forming a thick tissue capsule around the tubing or boiled clot. They contained contractile filaments and stained with antibodies to alpha-smooth muscle actin but no longer stained for Ly5.2. A separate set of female C57BL/6 Ly5.2 mice were X-irradiated to destroy bone marrow then immediately transfused with 10(6) nucleated bone marrow cells taken from the femur and tibia of a congenic strain of male mice expressing the Ly5.1 allele. Eight of the female mice with successful engraftment (80-99%) had silastic tubing implanted in the peritoneal cavity. After 14 days, in situ hybridization with Y chromosome probe confirmed the male donor, and thus bone marrow, origin of the elongated cells that formed the capsule. In vitro studies showed that cells of the murine macrophage cell lines RAW 264.7 and J774 express alpha-smooth muscle actin after exposure to the cytokine gamma-interferon in vitro. These data show that bone marrow-derived cells can differentiate into smooth muscle-like cells and raises the possibility that blood-derived cells may contribute to the development of fibro-proliferative vascular diseases such as atherosclerosis.

Heterogeneous distribution of isoactins in cultured vascular smooth muscle cells does not reflect segregation of contractile and cytoskeletal domains.

We have previously demonstrated that alpha-smooth muscle (alpha-SM) actin is predominantly distributed in the central region and beta-non-muscle (beta-NM) actin in the periphery of cultured rabbit aortic smooth muscle cells (SMCs). To determine whether this reflects a special form of segregation of contractile and cytoskeletal components in SMCs, this study systematically investigated the distribution relationship of structural proteins using high-resolution confocal laser scanning fluorescent microscopy. Not only isoactins but also smooth muscle myosin heavy chain, alpha-actinin, vinculin, and vimentin were heterogeneously distributed in the cultured SMCs. The predominant distribution of beta-NM actin in the cell periphery was associated with densely distributed vinculin plaques and disrupted or striated myosin and alpha-actinin aggregates, which may reflect a process of stress fiber assembly during cell spreading and focal adhesion formation. The high-level labeling of alpha-SM actin in the central portion of stress fibers was related to continuous myosin and punctate alpha-actinin distribution, which may represent the maturation of the fibrillar structures. The findings also suggest that the stress fibers, in which actin and myosin filaments organize into sarcomere-like units with alpha-actinin-rich dense bodies analogous to Z-lines, are the contractile structures of cultured SMCs that link to the network of vimentin-containing intermediate filaments through the dense bodies and dense plaques.

Oral administration of inducible nitric oxide synthase inhibitors reduces nitric oxide synthesis but has no effect on the severity of experimental colitis.

BACKGROUND: Increased concentrations of nitrate and nitrite (the breakdown products of nitric oxide) in the serum and faeces of patients with inflammatory bowel disease (IBD) suggests that increased synthesis of nitric oxide occurs in IBD. The aim of this study was to assess aminoguanidine (AMG), a selective inhibitor of inducible nitric oxide synthase, with regard to its effectiveness as a nitric oxide inhibitor and as a modulator of inflammation in trinitrobenzene sulfonic acid (TNBS)-induced colitis. MATERIALS AND METHODS: Colitis was induced in Wistar rats. Selective (AMG) and non-selective (1-nitroso-arginine methyl ester (1-NAME)) inhibitors of nitric oxide synthase were given in the drinking water. Colonic citrulline and arginine concentrations were assessed using high-performance liquid chromatography. The severity of colitis was assessed by a macroscopic scoring system. RESULTS: Both 1-NAME and AMG successfully reduced nitric oxide synthesis. There was no evidence of substrate depletion in the colonic wall. Neither of the agents reduced the severity of colonic inflammation. CONCLUSIONS: Oral administration of nitric oxide synthase inhibitors reduced nitric oxide synthesis in the colonic wall. This study does not provide evidence to support a role for nitric oxide in the pathogenesis of colonic inflammation in TNBS colitis.

The effect of environmental cues on the differentiation of myofibroblasts in peritoneal granulation tissue.

This study investigated the effect of haemodynamic stress, active stretch, and neuronal input on the differentiation of myofibroblasts in peritoneal granulation tissue. Lengths of silastic tubing (10 mm long x 3 mm diameter) were placed in the peritoneal cavity of the rat. By 2 weeks, a capsule of granulation tissue had formed around the tubing. This capsule consisted of several layers of myofibroblasts and the matrix that they had produced, overlaid by a single layer of mesothelial cells. The silastic tubing was removed and at the same time, the living tube of tissue was everted so that the mesothelium now lined its inner surface. To examine the effect of haemodynamic factors on myofibroblast differentiation, the 10 mm long tubes of mesothelial-lined granulation tissue were transplanted into the severed abdominal aorta of the same rat in which the granulation tissue was grown. End-to-end anastomoses were performed to extend the existing aorta. At 1, 2, and 3 months post-transplantation, the grafts were removed and a progressive increase in the percent volume fraction of myofilaments (% V(v)myo) was observed (from 35.7+/-1.6% to 58.7 3+/-1.4%; p<0.05). To determine whether the active stretching that occurs in vivo could account for differentiation of the constituent myofibroblasts, tubes of granulation tissue were placed into a mechanical device in which they underwent continuous stretching of 5-10% elongation from the resting position at 50 cycles per minute for 3, 24 or 72 h. This caused a significant (p<0. 05) increase in %V(v)myo after 72 h. Granulation tissue was also transplanted into the rat anterior eye chamber, where it became surrounded by adrenergic nerves supplying the host iris. Two months after implantation, there was no significant change in the %V(v)myo of the myofibroblasts (35.7+/-1.6% to 33.3+/-2.7%). These studies show that myofibroblasts of the granulation tissue encapsulating free-floating foreign bodies in the peritoneal cavity further differentiate towards a smooth muscle phenotype when transplanted into a smooth muscle environment, namely the abdominal aorta. Similar changes are seen when the granulation tissue is subjected to active, intermittent stretch in vitro, while the presence of nerves has no effect.