Daniel A. Martinez

Localization and early time course of TGF-β1 mRNA expression in dystrophic muscle

Fibrosis is a common pathological feature observed in muscle from patients with Duchenne muscular dystrophy (DMD). In the dystrophic (mdx) mouse model of DMD, the diaphragm is more severely affected than other skeletal muscles. The level of transforming growth factor‐beta1 (TGF‐β1), an inflammatory cytokine, is significantly elevated in mdx diaphragm. However, little is known about the onset of TGF‐β1 messenger ribonucleic acid (mRNA) expression, or which cells express the mRNA. In this study, we characterized the location and time course of expression of TGF‐β1 mRNA in diaphragm from mdx mice. TGF‐β1 mRNA was significantly elevated in mdx diaphragm at 6 and 9 but not 12 weeks of age, and these changes corresponded with changes in type I collagen mRNA and hydroxyproline concentration. Mononucleated cells localized to areas of fiber necrosis highly expressed the TGF‐β1 transcript in mdx diaphragm. Neutralization of TGF‐β1 by decorin administration resulted in a 40% reduction in the level of diaphragm muscle type I collagen mRNA. These findings support a role for TGF‐β1 during the early stages of fibrogenesis in dystrophic diaphragm muscle. Therapeutic interventions aimed at neutralizing this cytokine may be beneficial in slowing the development of fibrosis in DMD. Muscle Nerve, 2004

Exercise training reduces fibrosis and matrix metalloproteinase dysregulation in the aging rat heart

Aging impairs function in the nonischemic heart and is associated with mechanical remodeling. This process includes accumulation of collagen (i.e., fibrosis) and dysregulation of active matrix metalloproteinases (MMPs). Exercise training (ET) improves cardiac function, but the pathways of protection remain poorly understood. Young (3 mo) and old (31 mo) FBNF1 rats were assigned into sedentary and exercise groups, with ET group rats training on a treadmill 45 min/d, 5 d/wk for 12 wk. Nonlinear optical microscopy (NLOM), histology, immunohistochemistry (IHC), and Western blot analyses were performed on the left ventricle and septum. NLOM, IHC, and histological imaging revealed that ET reduced age‐associated elevation of collagen type I fibers. Active MMP‐1, active MMP‐2, and MMP‐14 in the ECM fraction of the left ventricle were reduced by aging, an effect abrogated by ET. Tissue inhibitor of MMP (TIMP‐1) was elevated with age but protected by ET. Transforming growth factor‐β (TGF‐β), upstream regulator of TIMP‐1, increased with age but was attenuated by ET. Therefore, exercise training could protect the aging heart against dysregulation of MMPs and fibrosis by suppressing elevation of TIMP‐1 and TGF‐β.—Kwak, H.‐B., Kim, J.‐H., Joshi, K., Yeh, A., Martinez, D. A., Lawler, J. M. Exercise training reduces fibrosis and matrix metalloproteinase dysregulation in the aging rat heart. FASEB J. 25, 1106–1117 (2011). www.fasebj.org

Effects of spaceflight on rat humerus geometry, biomechanics, and biochemistry.

The effects of a 12.5‐day spaceflight (Cosmos 1887 biosatellite) on the geometric, biomechanical, and biochemical characteristics of humeri of male specific pathogen‐free rats were examined. Humeri of age‐matched basal control, synchronous control, and vivarium control rats were contrasted with the flight bones to examine the influence of growth and space environment on bone development. Lack of humerus longitudinal growth occurred during the 12.5 days in spaceflight. In addition, the normal mid‐diaphysial periosteal appositional growth was affected; compared with their controls, the spaceflight humeri had less cortical cross‐sectional area, smaller periosteal circumferences, smaller anterior‐posterior periosteal diameters, and smaller second moments of area with respect to the bending and nonbending axes. The flexural rigidity of the flight humeri was comparable to that of the younger basal control rats and significantly less than that of the synchronous and vivarium controls; the elastic moduli of all four groups, nonetheless, were not significantly different. Generally, the matrix biochemistry of the mid‐diaphysial cross sections showed no differences among groups. Thus, the spaceflight differences in humeral mechanical strength and flexural rigidity were probably a result of the differences in humeral geometry rather than material properties.—Vailas, A. C.; Zernicke, R. F.; Grindeland, R. E., Kaplansky, A.; Durnova, G. N., Li, K.‐C.; and Martinez, D. A. Effects of spaceflight on rat humerus geometry, biomechanics, and biochemistry. FASEB J. 4: 47‐54; 1990.

Mechanical and biochemical analyses of tibial compartment fascia in chronic compartment syndrome

Increases in compartment pressure associated with chronic compartment syndrome (CCS) may be due to changes in the mechanical properties and/or thickness of fascia (4,22). To explore this possibility, we compared the mechanical and biochemical characteristics (stiffness, thickness, time-dependent response, collagen content, and collagen crosslinking) of fascia from patients with symptomatic anterior compartment syndrome to fascia from adjacent collateral compartments. We tested 43 specimens harvested from 20 individuals during surgical fasciectomy. Properties of normal (lateral)-compartment (NC) and pathological (anterior)-compartment (PC) fascia were mechanically tested in the axial and transverse directions forming four groups. An external control group (EX) of six specimens of anterior and lateral-compartment fascia harvested from amputated legs was also included in the study. PC fascia was found to be thicker and structurally stiffer (elastic modulus times thickness) in the axial direction than was NC fascia (p≤0.05). No significant differences were found between NC and PC time-dependent response, although significant differences between percent relaxation in the pooled axial and transverse direction specimens were observed. No differences were found in the collagen content, as measured by hydroxyproline (Hyp) concentration, between NC and PC fascia. PC fascia was found to have less collagen crosslinking by hydroxylyslpyridinoline (HP) concentration. In conclusion, although this study does not elucidate etiological factors in CCS, the changes found in PC fascia suggest that fascial mechanical properties contribute to the pathology.

Systemic administration of IGF-I enhances healing in collagenous extracellular matrices: evaluation of loaded and unloaded ligaments

BackgroundInsulin-like growth factor-I (IGF-I) plays a crucial role in wound healing and tissue repair. We tested the hypotheses that systemic administration of IGF-I, or growth hormone (GH), or both (GH+IGF-I) would improve healing in collagenous connective tissue, such as ligament. These hypotheses were examined in rats that were allowed unrestricted activity after injury and in animals that were subjected to hindlimb disuse. Male rats were assigned to three groups: ambulatory sham-control, ambulatory-healing, and hindlimb unloaded-healing. Ambulatory and hindlimb unloaded animals underwent surgical disruption of their knee medial collateral ligaments (MCLs), while sham surgeries were performed on control animals. Healing animals subcutaneously received systemic doses of either saline, GH, IGF-I, or GH+IGF-I. After 3 weeks, mechanical properties, cell and matrix morphology, and biochemical composition were examined in control and healing ligaments.ResultsTissues from ambulatory animals receiving only saline had significantly greater strength than tissue from saline receiving hindlimb unloaded animals. Addition of IGF-I significantly improved maximum force and ultimate stress in tissues from both ambulatory and hindlimb unloaded animals with significant increases in matrix organization and type-I collagen expression. Addition of GH alone did not have a significant effect on either group, while addition of GH+IGF-I significantly improved force, stress, and modulus values in MCLs from hindlimb unloaded animals. Force, stress, and modulus values in tissues from hindlimb unloaded animals receiving IGF-I or GH+IGF-I exceeded (or were equivalent to) values in tissues from ambulatory animals receiving only saline with greatly improved structural organization and significantly increased type-I collagen expression. Furthermore, levels of IGF-receptor were significantly increased in tissues from hindlimb unloaded animals treated with IGF-I.ConclusionThese results support two of our hypotheses that systemic administration of IGF-I or GH+IGF-I improve healing in collagenous tissue. Systemic administration of IGF-I improves healing in collagenous extracellular matrices from loaded and unloaded tissues. Growth hormone alone did not result in any significant improvement contrary to our hypothesis, while GH + IGF-I produced remarkable improvement in hindlimb unloaded animals.

Extracellular matrix maturation in the left ventricle of normal and diabetic swine

The main objective of this study is to determine the transmural distribution of extracellular matrix (ECM) collagen and maturation in non-diabetic and diabetic hearts. The Yucatan miniature swine heart ECM was analyzed in eight streptozotocin (STZ) induced diabetic pigs (Diabetic-Swine) and age matched normal control pigs (Nondiabetic-Swine). After 12 weeks of STZ induced diabetes, transmural biopsies were obtained from the left ventricular free wall divided into subendocardial, mid- and subepicardial layers. Collagen concentration and maturation were measured by RP-HPLC determination of hydroxyproline (Hyp) and content of hydroxylysylpyridinoline (HP) cross-links, respectively. Results showed a significant elevation in arterial glucose (P<0.05) and reduction in arterial plasma insulin levels in the Diabetic-Swine. Hyp concentration was significantly greater (P<0.05) in the subendocardial layers in both the Diabetic and Nondiabetic animals. The HP cross-link content was significantly greater (17%) in the Diabetic-swine subendocardial layer compared to Nondiabetic-Swine (P<0.05), but not in other layers. In summary, the accumulation and/or increase in HP cross-link content in the Diabetic-Swine subendocardial layer suggests that myocardial fibrosis may be greater in this specific region.

Impact of TNF‐α Blockade on TGF‐β1 and type I collagen mRNA expression in dystrophic muscle

Dystrophin-deficient diaphragm muscle generally follows a pathological cascade of muscle damage, necrosis, and fibrosis,13 leading to increased weakness and stiffness.13 The precise mechanisms leading to fibrosis in dystrophic muscle are not known but likely involve the action of several inflammatory cytokines such as tumor necrosis factor– (TNF) and transforming growth factor– 1 (TGF1). TGF1 is upregulated in dystrophic skeletal muscle2 and is known to influence collagen metabolism.7 However, it is unknown whether TGF1 is solely responsible for development of muscle fibrosis or whether it works in concert with other cytokines. TNFis a proinflammatory cytokine produced by activated macrophages3 and T cells, and its level is increased in dystrophic muscle.10 By magnifying the cellular and mediator responses both locally and systemically, TNFmay play a critical role during inflammation.1 Although TNFstimulates collagen metabolism in vitro, little is known about its role in the pathogenesis of fibrosis in dystrophic diaphragm muscle. We hypothesized that TNFblockade via Enbrel administration would significantly attenuate the expression of both TGF1 and type I collagen messenger RNA (mRNA) expression in diaphragm muscle from young mdx mice. Male control C57BL/10ScSn and mdx mice (Jackson Laboratories, Bar Harbor, Maine) were used in this study. Mice were provided with water and chow ad libitum and were housed in the Laboratory Animal Facility at the University at Buffalo, where all procedures were approved by the local Animal Care and Use Committee. Type I collagen mRNA and TGF1 were assessed in diaphragm muscle from 6-week-old control, untreated mdx, and Enbrel-treated mdx mice (n 5 per group). The Enbrel-treated mdx mice were given a daily dose of Enbrel (10 g/kg body weight i.p.) dissolved in phosphate-buffered saline (pH 7.4) for 10 consecutive days. Enbrel (Amgen, Thousand Oaks, CA) is a soluble receptor fusion protein that acts by binding TNF, thereby reducing the bioavailability of TNFmolecules to TNFreceptors. Real-time quantitative polymerase chain reaction (PCR) was used to assess changes in TGF1, collagen type I (Col 1A2) mRNA. Total RNA was isolated using the Tri-spin method according to Reno et al.11 Total RNA was quantified by optical density at 260 nm. Total RNA was reversetranscribed using SuperScript II RNase H (Invitrogen Corp., Carlsbad, CA) according to instructions. The 5 -Taqman quantitative PCR assays were performed using an MX-4000 Real Time Q-PCR instrument (Stratagene Inc., La Jolla, CA). The sequences for forward and reverse primers and fluorescent reporter probes (5 FAM and 3 -BHQ-1 Black HoleTM quenchers) (BioSearch Technologies, Novato, CA) were designed using Beacon Designer 2 software (Premier Biosoft, Palo Alto, CA) spanning at least one exon–exon junction to eliminate genomic contamination. Standards were generated using synthetic-DNA amplicon oligonucleotides (BioSource Int., Camarillo, CA) spanning the entire amplification region. Standard curves [ R 0.995; efficiencies (slope), 3.2 to 3.3] were generated to determine copy number of gene targets using synthetic DNA aliquots (10 to 10 copies). Quantitative PCR reactions were performed under the following conditions: 12 min at 95°C followed by 40 cycles of two-step thermocycling (45 s at 94°C and 12 s at 60°C) using Hotstar Taq polymerase (Qiagen, Inc., Valencia, CA). Each sample was run in triplicate. Changes in mRNA levels were analyzed using a one-way analysis of variance with post hoc (Student–Newman–Keuls) analysis. Statistical significance was placed at P 0.05. Figure 1 illustrates the mRNA levels for TGF1 (Fig. 1A) and 2(I) collagen (Fig. 1B) in diaphragm muscle from the three groups of mice. Compared with controls, the mean TGF1 mRNA level was increased over threefold in untreated mdx mice (P 0.002). Diaphragm muscle from Enbrel-treated mdx mice had a significantly lower TGF1 mRNA level (P 0.001) than untreated mdx mice, whereas the Enbrel-treated group did not significantly differ from control. Type I collagen gene expression was elevated over sevenfold in mdx diaphragm relative to control (P 0.001). In contrast, treating mdx mice with Enbrel significantly reduced diaphragm muscle type I collagen mRNA expression (P 0.001) compared with untreated mdx mice—no difference existed between control mice and Enbrel-treated mdx mice.

Elastic and physicochemical relationships within cortical bone

The purpose of this study was to examine the relationships that exist between the elastic properties and the physicochemical properties of cortical bone in two groups of experimental animals. The animal model was the immature mutant dwarf rat, and the groups consisted of rats treated and not treated with recombinant human growth hormone (rhGH). The objective was to establish and broaden the quantifiable link between the three-dimensional form and function of bone beyond the typical unidirectional measures. This study was based on previously reported work that refined the ultrasonic elasticity technique for use with small specimens (<1.0 mm) and determined that the administration of rhGH can counter the degenerative effects produced by hormone-suppressed downregulation on the elastic and physicochemical characteristics of cortical bone. Ultrasonic wave propagation and density measurements were used previously to determine the three-dimensional (orthotropic) material properties of rat femoral cortical bone. X-ray powder diffraction, microscopic, morphometric, and biochemical analysis techniques have been used to describe physicochemical properties, including mineral crystal size, cortical porosity, mineral and nonmineral content, and microstructural characteristics. In this study, mathematical relationships between the local physicochemical (independent variable) and elastic (dependent variable) properties were formulated via linear and nonlinear regression analyses. In general, apparent density was found to have the highest level of correlation with most of the longitudinal and shear moduli (R(2) = 0.300 to 0.800). Concomitantly, mineral crystal width and cortical porosity offered the best correlations with the Poissons ratios (R(2) up to 0.600). Wilcoxon t tests verified a significant decrease in the elastic properties in dwarf rat cortical bone after rhGH treatments (p < 0.05). Physicochemical measures of bone quality (density, crystal size) generally decreased while measures of bone quantity (cortical area, moments of inertia) generally increased (p < 0.05) after rhGH treatments. Some mineral and nonmineral properties were unchanged. This study presents a quantifiable link between cortical bone elasticity and its composite construction as measured across two dramatically different experimental groups.

EUK-134 ameliorates nNOSμ translocation and skeletal muscle fiber atrophy during short-term mechanical unloading

Reduced mechanical loading during bedrest, spaceflight, and casting, causes rapid morphological changes in skeletal muscle: fiber atrophy and reduction of slow-twitch fibers. An emerging signaling event in response to unloading is the translocation of neuronal nitric oxide synthase (nNOSμ) from the sarcolemma to the cytosol. We used EUK-134, a cell-permeable mimetic of superoxide dismutase and catalase, to test the role of redox signaling in nNOSμ translocation and muscle fiber atrophy as a result of short-term (54 h) hindlimb unloading. Fischer-344 rats were divided into ambulatory control, hindlimb-unloaded (HU), and hindlimb-unloaded + EUK-134 (HU-EUK) groups. EUK-134 mitigated the unloading-induced phenotype, including muscle fiber atrophy and muscle fiber-type shift from slow to fast. nNOSμ immunolocalization at the sarcolemma of the soleus was reduced with HU, while nNOSμ protein content in the cytosol increased with unloading. Translocation of nNOS from the sarcolemma to cytosol was virtually abolished by EUK-134. EUK-134 also mitigated dephosphorylation at Thr-32 of FoxO3a during HU. Hindlimb unloading elevated oxidative stress (4-hydroxynonenal) and increased sarcolemmal localization of Nox2 subunits gp91phox (Nox2) and p47phox, effects normalized by EUK-134. Thus, our findings are consistent with the hypothesis that oxidative stress triggers nNOSμ translocation from the sarcolemma and FoxO3a dephosphorylation as an early event during mechanical unloading. Thus, redox signaling may serve as a biological switch for nNOS to initiate morphological changes in skeletal muscle fibers.

Effect of a growth hormone treatment on bone orthotropic elasticity in dwarf rats.

A refinement of the current ultrasonic elasticity technique was used to measure the orthotropic elastic properties of rat cortical bone as well as to quantify changes in elastic properties, density, and porosity of the dwarf rat cortex after a treatment with recombinant human growth hormone (rhGH). The ultrasonic elasticity technique was refined via optimized signal management of high-frequency wave propagation through cubic cortical specimens. Twenty dwarf rats (37 days old) were randomly assigned to two groups (10 rats each). The dwarf rat model (5–10% of normal GH) was given subcutaneous injections of either rhGH or saline over a 14-day treatment period. Density was measured using Archimedes’ technique. Porosity and other microstructural characteristics were also explored via scanning electron microscopy and image analysis. Statistical tests verified significant decreases in corticla orthotropic Young’s (−26.7%) and shear (−16.7%) moduli and density (−2.42%) concomitant with an increase in porosity (+125%) after rhGH treatments to the dwarf model (p<0.05). A change in material symmetry from orthotropy toward planar isotropy within the radial-circumferential plane after GH treatments was also noted. These results demonstrate some alteration in bone properties at this time interval. Structural implications of these changes throughout physiological loading regimens should be explored.