John A. Thompson

Decreased zinc affinity of amyotrophic lateral sclerosis-associated superoxide dismutase mutants leads to enhanced catalysis of tyrosine nitration by peroxynitrite

Abstract: Mutations to Cu/Zn superoxide dismutase (SOD) linked to familial amyotrophic lateral sclerosis (ALS) enhance an unknown toxic reaction that leads to the selective degeneration of motor neurons. However, the question of how >50 different missense mutations produce a common toxic phenotype remains perplexing. We found that the zinc affinity of four ALS‐associated SOD mutants was decreased up to 30‐fold compared to wild‐type SOD but that both mutants and wild‐type SOD retained copper with similar affinity. Neurofilament‐L (NF‐L), one of the most abundant proteins in motor neurons, bound multiple zinc atoms with sufficient affinity to potentially remove zinc from both wild‐type and mutant SOD while having a lower affinity for copper. The loss of zinc from wild‐type SOD approximately doubled its efficiency for catalyzing peroxynitrite‐mediated tyrosine nitration, suggesting that one gained function by SOD in ALS may be an indirect consequence of zinc loss. Nitration of protein‐bound tyrosines is a permanent modification that can adversely affect protein function. Thus, the toxicity of ALS‐associated SOD mutants may be related to enhanced catalysis of protein nitration subsequent to zinc loss. By acting as a high‐capacity zinc sink, NF‐L could foster the formation of zinc‐deficient SOD within motor neurons.

Peroxynitrite-induced cytotoxicity in PC12 cells : evidence for an apoptotic mechanism differentially modulated by neurotrophic factors

Abstract: Peroxynitrite is a powerful oxidant formed by the near‐diffusion‐limited reaction of nitric oxide with superoxide. Large doses of peroxynitrite (>2 mM) resulted in rapid cell swelling and necrosis of undifferentiated PC12 cells. However, brief exposure to lower concentrations of peroxynitrite (EC50 = 850 µM) initially (3–4 h) caused minimal damage to low‐density cultures. By 8 h, cytoplasmic shrinkage with nuclear condensation and fragmentation became increasingly evident. After 24 h, 36% of peroxynitrite‐treated cells demonstrated these features associated with apoptosis. In addition, 46% of peroxynitrite‐treated cells demonstrated DNA fragmentation (by terminal‐deoxynucleotide transferase‐mediated dUTP‐digoxigenin nick end‐labeling) after 7 h, which was inhibited by posttreatment with the endonuclease inhibitor aurintricarboxylic acid. Serum starvation also resulted in apoptosis in control cells (23%), the percentage of which was not altered significantly by peroxynitrite treatment. Although peroxynitrite is known to be toxic to cells, the present study provides a first indication that peroxynitrite induces apoptosis. Furthermore, pretreatment of cells with nerve growth factor or insulin, but not epidermal growth factor, was protective against peroxynitrite‐induced apoptosis. However, both acidic and basic fibroblast growth factors greatly increased peroxynitrite‐initiated apoptosis, to 63 and 70%, respectively. Thus, specific trophic factors demonstrate differential regulation of peroxynitrite‐induced apoptosis in vitro.

Direct In Vivo Evidence Demonstrating Neointimal Migration of Adventitial Fibroblasts After Balloon Injury of Rat Carotid Arteries

BACKGROUND Clinical and experimental evidence suggest that the adventitia participates in the response to endoluminal vascular injury. The current study used a direct approach to test the hypothesis that, after balloon injury of the rat carotid artery, adventitial fibroblasts migrate in a luminal direction and contribute to neointima formation. METHODS AND RESULTS Primary syngeneic adventitial fibroblasts were stably transduced with retroviral particles coordinating expression of beta-galactosidase (LacZ) and introduced into the adventitia of right carotid arteries of rats immediately after balloon injury. At defined times after injury and fibroblast implantation, rats were euthanized, and arterial tissue was examined for detection of LacZ mRNA (reverse transcription polymerase chain reaction), DNA (polymerase chain reaction), and in situ enzymatic activity. LacZ expression was detected in the media 5 days postinjury and in both media and neointima at 7, 10, and 14 days postinjury. LacZ was undetectable in injured vessels that had not been seeded with transduced fibroblasts and was restricted to the adventitia in seeded vessels that were not injured. CONCLUSIONS These observations provide direct demonstration of adventitial fibroblast migration into neointima of arteries after endoluminal injury.

Mitochondrial tyrosine nitration precedes chronic allograft nephropathy

Endogenous tyrosine nitration and inactivation of manganese superoxide dismutase (MnSOD) has previously been reported to occur during end-stage human renal allograft rejection. In order to determine whether nitration and inactivation of this critical mitochondrial protein might play a contributory role in the onset of transplant rejection, we employed a rodent model of Chronic Allograft Nephropathy (or CAN). Using this model we followed kidney function from 2-52 weeks post-transplant and correlated graft function with levels of nitration in the renal allograft. Tyrosine nitration of both glomerular and tubular structures occurred at 2 weeks post-transplant. At later times (16 weeks) post-transplant, tyrosine nitration appeared to be confined to tubular structures; however glomerular nitration returned at 52 weeks post-transplant. Interestingly, nitration and inactivation of MnSOD occurs prior to the onset of renal dysfunction in this rat model of chronic allograft nephropathy (2 weeks versus 16 weeks post-transplant). Furthermore, we have identified an additional mitochondrial protein, cytochrome c, as being endogenously nitrated during chronic rejection. The kinetics of cytochrome c nitration lagged behind MnSOD nitration and inactivation (4 weeks compared to 2 weeks); suggesting that loss of MnSOD activity likely contributes to elevation of the nitrating species and further nitration of other targets.

Serum-starvation induces the extracellular appearance of FGF-1.

Autocrine/paracrine stimulation of cell growth by members of the fibroblast growth factor (FGF) family of polypeptides is dependent upon extracellular interactions with specific high affinity receptors at the cell surface. Acidic FGF (FGF-1) lacks a classical signal sequence for secretion, suggesting that intrinsic levels of this mitogen may not stimulate cell growth and utilizes a non-classical pathway to gain access to the extracellular compartment. To evaluate the biological potential of intracellular FGF-1 more rigorously, human cDNA sequences for the growth factor were introduced into primary murine embryonic fibroblasts using retrovirally mediated gene transfer. Heparin affinity, Western analysis, mitogenic assays, in situ immunohistochemical techniques, induction of tyrosine phosphorylation and antibody inhibition studies were used to demonstrate functionality of the FGF-1 transgene in this experimental model. Under normal culture conditions, cells constitutively expressing intracellular FGF-1 exhibited a slight growth advantage. In contrast, when maintained in reduced serum, these cells adopted a transformed phenotype and demonstrated an enhanced growth potential, induction of FGF-specific phosphotyrosyl proteins and the nuclear association of the growth factor. Analysis of the conditioned media from these stressed cells indicated that serum starvation induces the secretion of FGF-1 as latent high molecular mass complexes requiring reducing agents to activate its full biological potential.

Estrogen attenuates the adventitial contribution to neointima formation in injured rat carotid arteries.

OBJECTIVE This study tested, in ovariectomized rats, whether (1) adventitial activation plays a role in the vascular injury response, and (2) inhibition of adventitial activation and the subsequent wave of cell proliferation moving from adventitia to neointima contributes to the estrogen-induced attenuation of neointima formation in balloon injured carotid arteries. METHODS Ovariectomized Sprague-Dawley rats were treated with either 17 beta-estradiol or vehicle beginning 72 h prior to balloon injury of the right common carotid artery and were sacrificed at 0, 3, 7, 14 and 28 days after injury. BrdU was administered 18 h and 12 h prior to sacrifice in order to quantitate mitotic activity in adventitia, media and neointima of the damaged vessel at specified times post injury. RESULTS Adventitial activation, evidenced by positive BrdU staining, was evident on the day of injury, peaked on day 3 and was resolved by day 7, thus preceding neointima formation. Numbers of BrdU labeled cells in adventitia on day 3 were significantly reduced in estrogen treated rats compared to controls. BrdU labeled cells were undetectable in media on the day of injury, appeared at day 3 and disappeared by day 14. Neointima appeared at day 7 and increased in area throughout the period of observation. Neointimal area and numbers of BrdU labeled cells in neointima were significantly reduced in estrogen treated rats compared to controls. These findings suggest that there is a wave of cell proliferation moving in an adventitia-to-lumen direction following endoluminal injury of the rat carotid artery and that estrogen modulates this proliferative response to injury. CONCLUSION These results support the hypothesis that adventitial activation contributes to the vascular injury response and that estrogen reduces this contribution.

Estrogen Inhibits Vascular Smooth Muscle Cell–Dependent Adventitial Fibroblast Migration In Vitro

BACKGROUND Mounting experimental evidence suggests that estrogen treatment protects against neointima formation in response to vascular injury in vivo. Previous studies have suggested that this process includes the activation and migration of adventitial fibroblasts. The present in vitro study was designed to establish a mechanism whereby estrogen attenuates migration of adventitial fibroblasts. METHDS AND RESULTS: Primary cultures of vascular smooth muscle cells (VSMCs) and adventitial fibroblasts were derived from female Sprague-Dawley rats. Reverse transcriptase-polymerase chain reaction and Western blotting were used to determine that expression of the estrogen receptor (ER) was restricted to early-passage VSMCs. Migration of transduced (retrovirally mediated) fibroblasts was determined by counting the number of blue lacZ-expressing cells attached to Boyden-type chambers preconditioned under defined experimental conditions. Compared with growth medium alone, chambers treated with medium conditioned by VSMCs demonstrated a 2-fold increase in fibroblast migration, suggesting that VSMCs release soluble factor(s) competent to bind the Transwell membrane and promote fibroblast migration. In contrast, treatment of VSMCs with 17beta-estradiol (10(-9) to 10(-7) mol/L) before preconditioning of the chamber induced a dose-dependent inhibition of fibroblast migration. Cotreatment of VSMCs with 17beta-estradiol and the ER antagonist ICI-182780 (10(-7) mol/L) blocked the inhibitory effect of estrogen on fibroblast migration. CONCLUSIONS These observations suggest a novel mechanism of hormonal vasoprotection by which estrogen directly modulates VSMC expression of factor(s) controlling migration of adventitial fibroblasts via an ER-dependent mechanism.

Fibrin Scaffold as an Effective Vehicle for the Delivery of Acidic Fibroblast Growth Factor (FGF-1):

The effect of wound healing by fibrin and acidic fibroblast growth factor (FGF-1) in an in vivo model was evaluated in this study. Four full-thickness wounds were made on the dorsum of each rabbit (n = 5). Each of these wounds had different treatment groups: control, topical FGF-1 (100 μg/9 cm2), fibrin (2.0 mL at 60 mg/mL fibrinogen), and FGF-1 (100 gg/9 cm2)/fibrin. The animals were sacrificed at the end of 2 weeks. Histomorphometric analysis and mechanical testing were conducted to assess the healing response. FGF-1/fibrin treatment improved the mechanical properties of the healed tissue. Fibrin scaffold exhibited the desired tissue response, as demonstrated by the lack of inflammation, and was deemed an effective carrier for FGF-1.

Fibroblast Growth Factor Receptor-1 Signaling Induces Osteopontin Expression and Vascular Smooth Muscle Cell–Dependent Adventitial Fibroblast Migration In Vitro

Background—Increased expression of osteopontin (OPN), fibroblast growth factors (FGFs), and their type-1 receptor (FGFR-1) is associated with neointima formation and atherosclerosis. This study tested the hypothesis that ligand activation of FGFR-1 stimulates OPN expression in rat aortic smooth muscle cells (RASMCs), explored the signaling pathway involved, and assessed the functional consequences of activating this pathway on adventitial fibroblast (AF) migration in vitro. Methods and Results—Exogenous FGF-1 stimulated expression of OPN mRNA and protein in RASMCs in vitro in a dose- and time-dependent manner. OPN mRNA induction by FGF-1 was completely inhibited by either actinomycin D or cycloheximide, selective inhibitors of RNA polymerase and protein synthesis, respectively. OPN mRNA induction by FGF-1 was attenuated by PD 166866, a highly selective and potent FGFR-1 tyrosine kinase inhibitor. Addition of either PP2 or PD98059, specific inhibitors of Src and mitogen-activated extracellular signal–regulated kinase (MEK)/mitogen-activated protein (MAP) kinases, respectively, attenuated FGF-1–stimulated OPN mRNA expression. FGF-1 treatment of RASMCs enhanced RASMC-conditioned medium-stimulated AF migration; this effect was inhibited by pretreatment of RASMCs with either PD166866 or PP2. Immunodepletion of OPN from RASMC-conditioned medium inhibited both basal and FGF-1–stimulated AF migration. Conclusions—This in vitro study provided a first indication that ligand-activated FGFR-1 plays a significant role in upregulation of OPN expression at the transcriptional level via signaling to Src/MEK/MAP kinases in RASMCs and that this pathway is functionally significant in mediating AF migration via stimulation of OPN expression.

Estrogen Attenuates Integrin-β3–Dependent Adventitial Fibroblast Migration After Inhibition of Osteopontin Production in Vascular Smooth Muscle Cells

BACKGROUND Previous in vitro studies have suggested that estrogen attenuates the vascular injury response by modulating vascular smooth muscle cell (VSMC) expression of soluble factor(s) directing migration of adventitial fibroblasts. Previous in vivo studies have established a role for osteopontin (OPN) and its integrin receptors after vascular injury. In this study, we examined OPN expression in activated VSMCs, its modulation by estrogen, and its effects on adventitial fibroblast migration. In addition, the relative functional roles of beta(1)- and beta(3)-integrin-matrix interactions were examined. METHODS AND RESULTS Primary cultures of VSMCs and adventitial fibroblasts were derived from female Sprague-Dawley rats. Serum-activated VSMCs expressed high levels of OPN mRNA and secreted protein that was effectively inhibited by estrogen treatment (10(-7) mol/L). Compared with VSMCs, fibroblasts expressed similar levels of integrins alphanu and beta(1) and higher levels of integrin-beta(3). Exogenous OPN (5.0 to 40 microg/mL) directed fibroblast migration in a dose-dependent fashion. Anti-beta(3)-integrin antibody (F11) pretreatment markedly inhibited adventitial fibroblast migration directed by exogenous OPN or VSMC-conditioned medium in a dose-dependent manner. In contrast, anti-beta(1)-integrin antibody (Ha2/5) did not affect fibroblast migration. Similarly, pretreatment with either linear or cyclic RGD peptides (10 to 1000 micromol/L) inhibited fibroblast migration directed by OPN or VSMC-conditioned medium in a dose-dependent manner. CONCLUSIONS These observations suggest that estrogen indirectly attenuates integrin-beta(3)-dependent adventitial fibroblast migration after inhibition of OPN expression in VSMCs.