Morphological Changes and Immunohistochemical Expression of RAGE and its Ligands in the Sciatic Nerve of Hyperglycemic Pig (Sus Scrofa)

The aim of our project was to study the effect of streptozotocin (STZ)—induced hyperglycemia on sciatic nerve morphology, blood plasma markers and immunohistochemical expression of RAGE (the Receptor for Advanced Glycation End-products), and its ligands—S100B and Carboxymethyl Lysine (CML)-advanced glycation endproduct (AGE) in the laboratory pig. Six months after STZ—injections, blood plasma measurements, morphometric analysis of sciatic nerve fiber density, immunofluorescent distribution of potential molecular neuropathy contributors, ELISA measurement of plasma AGE level and HPLC analysis of sciatic nerve levels of one of the pre-AGE and the glycolysis intermediate products—methyl-glyoxal (MG) were performed. The results of our study revealed that STZ—injected animals displayed elevated levels of plasma glucose, gamma glutamyl transferase (GGT) and triglycerides. The sciatic nerve of STZ-injected pigs revealed significantly lower numbers of small-diameter myelinated fibers, higher immunoreactivity for RAGE and S100B and increased levels of MG as compared to control animals. Our results correspond to clinical findings in human patients with hyperglycemia/diabetes-evoked peripheral neuropathy and suggest that the domestic pig may be a suitable large animal model for the study of mechanisms underlying hyperglycemia-induced neurological complications in the peripheral nerve and may serve as a relevant model for the pre-clinical assessment of candidate drugs in neuropathy

Early Treatment with Fumagillin, an Inhibitor of Methionine Aminopeptidase-2, Prevents Pulmonary Hypertension in Monocrotaline-Injured Rats

Pulmonary Hypertension (PH) is a pathophysiologic condition characterized by hypoxemia and right ventricular strain. Proliferation of fibroblasts, smooth muscle cells, and endothelial cells is central to the pathology of PH in animal models and in humans. Methionine aminopeptidase-2 (MetAP2) regulates proliferation in a variety of cell types including endothelial cells, smooth muscle cells, and fibroblasts. MetAP2 is inhibited irreversibly by the angiogenesis inhibitor fumagillin. We have previously found that inhibition of MetAP2 with fumagillin in bleomycin-injured mice decreased pulmonary fibrosis by selectively decreasing the proliferation of lung myofibroblasts. In this study, we investigated the role of fumagillin as a potential therapy in experimental PH. In vivo, treatment of rats with fumagillin early after monocrotaline injury prevented PH and right ventricular remodeling by decreasing the thickness of the medial layer of the pulmonary arteries. Treatment with fumagillin beginning two weeks after monocrotaline injury did not prevent PH but was associated with decreased right ventricular mass and decreased cardiomyocyte hypertrophy, suggesting a direct effect of fumagillin on right ventricular remodeling. Incubation of rat pulmonary artery smooth muscle cells (RPASMC) with fumagillin and MetAP2-targeting siRNA inhibited proliferation of RPASMC in vitro. Platelet-derived growth factor, a growth factor that is important in the pathogenesis of PH and stimulates proliferation of fibroblasts and smooth muscle cells, strongly increased expression of MetP2. By immunohistochemistry, we found that MetAP2 was expressed in the lesions of human pulmonary arterial hypertension. We propose that fumagillin may be an effective adjunctive therapy for treating PH in patients

Fumagillin treatment decreases the number of inflammatory cells following monocrotaline injury.

<p>Immunohistochemistry was performed for common leukocyte antigen (CD45). CD45+ cells were quantified per high power field (hpf) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035388#s2" target="_blank">Materials and Methods</a>. MCT injury was associated with a significant decrease in the number of CD45+ cells in the lung. Early treatment with fumagillin decreased CD45+ cells in the lung by 25% (*<i>P</i><0.05 by ANOVA, n = 3–4 animals per group).</p

Quantification of Ki67 Staining in Rat Pulmonary Arteries.

<p>Immunohistochemistry for Ki67 was performed on rat lung sections from animals injured with MCT treated with or without fumagillin. Images of pulmonary arteries are shown (A) uninjured + vehicle (B) uninjured + fumagillin (C) MCT + vehicle and (D) MCT + fumagillin (Magnification ×400, bar = 20 µm). Black arrows point to Ki67+ nuclei in the medial layer of pulmonary arteries and yellow arrows point to Ki67+ nuclei in endothelial cells. Magnification ×400, inset line 20 mm. (E) Ki67+ nuclei in the medial layer (n = 4–6 per group) and (F) Ki67+ nuclei in endothelial cells of the pulmonary arteries (n = 4 per group) were quantified per hpf as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035388#s2" target="_blank">Materials and Methods</a>. MCT injury resulted in a nearly 15-fold increase in the number of Ki67+ nuclei in medial cells per vessel per hpf in the MCT-injured vehicle-treated lung. The increase in the number of Ki67+ nuclei was decreased nearly 30% with fumagillin treatment, but this difference did not reach statistical significance. Similarly, MCT significantly increased the number of endothelial cells staining positively for Ki67 nearly tenfold. However, no differences in the number of Ki67+ nuclei were detected after fumagillin treatment.</p

Fumagillin Prevents Pulmonary Hypertension and Right Ventricular Hypertrophy in MCT-Injured Rats.

<p>Rats were injected with MCT and treated with fumagillin or vehicle control as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035388#s2" target="_blank">Materials and Methods</a>. After four weeks, animals were killed, and direct right ventricular pressure was measured (A). MCT injury significantly increased RV systolic BP, which is prevented by early, but not late, treatment with fumagillin (*<i>P</i><0.05, one-way ANOVA followed by Bonferroni's post hoc test). (B) Measurement of right ventricular mass normalized to left ventricular mass (RV/LV+S). Five weeks after MCT injury, hearts were excised, fixed in formalin, and the masses of the RV and the LV+septum were determined. MCT-injured animals treated with the vehicle exhibited a significant increase in RV mass, which was prevented in both early and late fumagillin-treated animals (*<i>P</i><0.05, ANOVA followed by Bonferroni's post-hoc test).</p

Fumagillin and Silencing of MetAP2 Expression Inhibit Proliferation of RPASMC <i>in vitro</i>.

<p>(A) Primary RPASMC were serum-starved in basal medium for 24 h to synchronize all cells in G0. Cells were then released from G0 by addition of 10% FBS supplemented with or without PDGF (10 ng/mL) or fumagillin (20 nM). Cells were fixed after 0, 1, 2, and 3 days of culture and then stained with SYBR Green as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035388#s2" target="_blank">Materials and Methods</a>. Fluorescent intensity, which correlates with relative cell number, was measured by a fluorimetric plate reader. Data represent mean + SEM, n = 4. At days 2 and 3, fumagillin significantly reduced cell number in cells incubated in the absence of PDGF (*<i>P</i><0.01) or in the presence of PDGF (**<i>P</i><0.001). (B–C) RPASMC were incubated in the absence of serum followed by addition of serum (B) or serum with PDGF (10 ng/mL) (C) with or without fumagillin at the indicated concentrations. Cells were incubated for an additional 48 h and pulsed with BrdU for the final 30 minutes then fixed. BrdU+ cells were quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035388#s2" target="_blank">Materials and Methods</a>. Data represent mean + SEM. Data analyzed by ANOVA followed by Tukey's multiple comparison test (*<i>P</i><0.05, fumagillin v vehicle AND fumagillin + PDGF v. vehicle + PDGF). (D) Immunoblotting for MetAP2 of RPASMC cultured in the presence of 10% serum and exposed to MetAP2-targeting siRNA (lanes 1 and 2) or non-targeting RNA oligonucleotides (lanes 3 and 4). Cells in lanes 1 and 3 were incubated with the vehicle control, and cells in lanes 2 and 4 were incubated with fumagillin 20 nM. Note the increased band intensity of MetAP2 in the fumagillin conditions. (E) Incorporation of BrdU in RPASMC during silencing of MetAP2 gene expression by siRNA. During suppression of MetAP2 gene expression, BrdU incorporation was decreased 27% compared to the non-targeting control (**<i>P</i><0.05, ANOVA followed by Neuman-Keuls post-hoc test compared to Non-targeting + VEH). In the presence of fumagillin, silencing of MetAP2 did not lead to additional inhibition of proliferation.</p

Treatment of monocrotaline-injured rats with fumagillin leads to improved right ventricular function.

<p>Rats were injected with MCT and treated with fumagillin or vehicle control beginning at 3 d or 14 d after MCT injury as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035388#s2" target="_blank">Materials and Methods</a>. At five weeks after MCT injury, animals were prepared for echocardiography and measurement of invasive hemodynamics as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035388#s2" target="_blank">Materials and Methods</a>: (A) right ventricular ejection fraction, (B) right ventricular minimum dP/dt, (C) right ventricular end-diastolic pressure, and (D) the ratio of right ventricular to pulmonary artery elastance, Ees/Ea. Note stepwise improvement in these hemodynamic parameters as fumagillin treatment is delivered late vs. early (*<i>P</i><0.05, MCT+early fumagillin v. MCT+vehicle, by t-test n = 4−7 animals per group).</p

Treatment with fumagillin decreases the number of apoptotic cells in rat hearts.

<p>Rats were injected with MCT and treated with fumagillin or vehicle control beginning at 14 d after MCT injury as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035388#s2" target="_blank">Materials and Methods</a>. At five weeks after MCT injury, animals were killed and the free wall of the right ventricles were prepared for TUNEL staining: (A) Uninjured + vehicle, (B) uninjured + fumagillin, (C) MCT + vehicle, week 2, (D) MCT + fumagillin early, and (E) MCT + fumagillin late (magnification ×400, bar = 20 µm). The number of TUNEL+ cells in the right ventricles were increased by MCT injury but attenuated by fumagillin treatment (F) (*<i>P</i><0.05, one-way ANOVA, followed by Bonferroni's post-hoc test, n = 3–5 animals per group).</p

Fumagillin Protects Rats from PA thickening in MCT-injured Rats.

<p>Rats were injected with MCT and underwent treatment with fumagillin or vehicle control as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035388#s2" target="_blank">Materials and Methods</a>. Animals were killed on day 35 after injury, and the lungs were processed for histology. Hematoxylin and eosin staining of lungs from (A) uninjured, vehicle-treated (B) uninjured, fumagillin-treated, (C) MCT-injured, vehicle treated, or (D) MCT-injured, early fumagillin treated. Arrows point to thickened pulmonary arteries (×100 magnification, scale bar = 50 µm). Inset images are 400× magnification and show immunohistochemistry for α-SMA (brown) and vWF (gray) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035388#s2" target="_blank">Materials and Methods</a>. Pulmonary artery thickness was measured by Movat staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035388#s2" target="_blank">Materials and Methods</a> (Representative images are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035388#pone.0035388.s001" target="_blank">Figure S1</a>) (E) All vessels <250 µm, (H) vessels <50 µm, (I) vessels <30 µm. *<i>P</i><0.05, n = 4–6 animals). Data were analyzed by one-way ANOVA followed by the Bonferroni post-hoc test.</p