The Selective Angiotensin II Type 2 Receptor Agonist Compound 21 Reduces Abdominal Adhesions in Mice.

BACKGROUND: Abdominal adhesions (AAs) are post-traumatic fibrous bands that connect visceral and/or peritoneal surfaces, leading to possible long-term complications. The effect of a novel antifibrotic selective angiotensin II type 2 receptor agonist, compound 21 (C21) on AA formation was assessed in a murine model. METHODS: Female BALB/c mice were laparotomized and the cecum and overlying parietal peritoneum abraded. C21 (10 µg/kg) or saline (vehicle) were administered orally or intraperitoneally daily. Mice were sacrificed 8 days after surgery, adhesions graded, and peritoneal fluid collected for transforming growth factor (TGF)-ß levels. Laparotomy incisions were excised for immunohistochemistry. In vitro, scratch assays were performed using primary parietal peritoneal fibroblasts and visceral mesothelial cells treated with C21 (10 µM), angiotensin II (1 µM), or both. Western blot analysis of primary cell lysates was performed for total and phosphorylated SMAD 2/3. RESULTS: Oral and intraperitoneal C21 reduced AA formation and TGF-ß levels in peritoneal fluid. Surgical incisions demonstrated decreased α-smooth muscle actin expression in C21-treated animals, but no difference in vascularity, macrophage infiltration, collagen I/III distribution and density, and dermal thickness. Migration and expression of phosphorylated SMAD 2/3 was reduced in parietal peritoneal fibroblasts and visceral mesothelial cells treated with C21. CONCLUSIONS: Local and systemic C21 administration reduced or completely prevented AA formation. These findings may be attributed to decreased intraperitoneal TGF-ß in vivo and decreased migration of peritoneal fibroblasts and visceral mesothelial cells. Importantly, C21 did not have histologically quantifiable effects on laparotomy wounds, suggesting C21 could reduce AA formation without compromising laparotomy healing.

The reversible P2Y12 inhibitor ticagrelor inhibits metastasis and improves survival in mouse models of cancer.

Tumor cells use activated platelets to promote their proliferation and metastatic potential. Because platelet activation is largely mediated through ADP engagement of purinergic P2Y12 receptors on platelets, we investigated the potential of the reversible P2Y12 inhibitor ticagrelor, a clinical agent used in the prevention of cardiovascular and cerebrovascular events, to inhibit tumor adhesion and metastasis. In B16-F10 melanoma intravenous and intrasplenic metastasis models, mice treated with a clinical dose of ticagrelor (10 mg/kg) exhibited marked reductions in lung (84%) and liver (86%) metastases. Furthermore, ticagrelor treatment improved survival compared to saline-treated animals. A similar effect was observed in a 4T1 breast cancer model, with reductions in lung (55%) and bone marrow (87%) metastases following ticagrelor treatment. In vitro, B16-F10 cells exhibited decreased interaction with platelets from ticagrelor-treated mice compared to saline-treated mice, an effect similar to that observed with blockade of glycoprotein IIbIIIa. Similarly, B16-F10 cells co-incubated with platelets from ticagrelor-treated mice exhibited reduced adhesion to endothelial monolayers compared to those co-incubated with platelets from saline-treated animals, an effect also observed in vivo. Interestingly, pretreatment of endothelial monolayers with ticagrelor did not result in reduced tumor cell adhesion. These findings support a role for P2Y12-mediated platelet activation in promoting metastases, and provide proof-of-concept for the clinical use of ticagrelor in the prevention of tumor metastasis.

Increase in endothelin B receptor expression in optic nerve astrocytes in endothelin-1 induced chronic experimental optic neuropathy.

The purpose of this study was to determine whether endothelin B (ETB) receptor levels in the optic nerve are related to retinal ganglion cell (RGC) loss in a model of chronic endothelin-1 (ET-1) induced optic neuropathy. RGCs of adult Brown Norway rats were first retrogradely labeled with fluorochrome from the superior colliculi. An osmotic minipump was surgically implanted 7 days later to deliver 10(-11) M (n = 9), 10(-9) M (n = 12) or 10(-7) M (n = 9) ET-1 to the retrobulbar optic nerve for 28 days. RGC survival was expressed as the ratio of RGC counts in experimental versus control eyes in wholemounted retinas. Optic nerves were used for either ETB western blot analysis (n = 24) or immunohistochemistry (n = 6) for ETB and glial fibrillary acidic protein (GFAP) to localize astrocytes. ETB expression was higher in the experimental nerve compared to the fellow untreated control nerve in 19 (79%) of the 24 animals with a mean increase of 16.7 +/- 4.5% in densitometric analyses of the immunoblots. Experimental nerves showed stronger labeling for both ETB and GFAP compared to control nerves. ETB-positive cells almost completely co-localized with GFAP-positive cells in both experimental and untreated control nerves, however, ETB expression was stronger in the astrocyte soma and proximal processes, while GFAP was expressed more strongly in the distal processes. There was a weak relationship between RGC loss and increase in ETB expression (r = -0.417, p = 0.076). There is an upregulation of ETB expression in optic nerve astrocytes in ET-1 induced chronic optic neuropathy causing RGC loss.

Induction of heat shock proteins 27 and 72 in retinal ganglion cells after acute pressure-induced ischaemia.

BACKGROUND: We wanted to investigate whether heat shock protein (HSP) 27 and HSP 72 are induced in retinal ganglion cells (RGCs) after acute intraocular pressure (IOP)-induced ischaemia. METHODS: Retinal ischaemia was induced by acutely increasing IOP to 100-110 mmHg for 30 or 90 min unilaterally in Sprague Dawley rats. A fluorescent tracer (fluorogold, FG) was applied to the superior colliculi to label RGCs. Twenty-four hours, 1 week or 2 weeks after of IOP elevation, rats were killed, RGCs counted, and immunohistochemical labelling of the retina was performed. HSP-positive RGCs were counted and normalized HSP RGC counts determined. RESULTS: The ratio of FG-positive labelled RGCs in the experimental to the contralateral eye as a marker of RGC survival remained unchanged after 30 min of ischaemia: 1.09 +/- 0.11 at 1 week, and 0.94 +/- 0.28 at 2 weeks. After 90 min of ischaemia RGC survival decreased to 0.19 +/- 0.14 at 1 week, and 0.20 +/- 0.14 at 2 weeks. After 30 min of ischaemia, the normalized HSP 27- and HSP 72-positive RGC count was detected at highest levels (HSP 27: 5.42 +/- 1.18; HSP 72: 12.23 +/- 1.24) at 2 weeks compared with controls,whereas after 90 min ischaemia it was detected at higher levels at 1 week (HSP 27: 52.63 +/- 3.65; HSP 72: 206.84 +/- 60.38), as well as at 2 weeks (HSP 27: 89.00 +/- 17.21; HSP 72: 191.00 +/- 50.05). CONCLUSION: These results demonstrate an enhanced induction of HSP 27 and HSP 72 after 90 min acute IOP-induced ischaemia. In contrast to 30 min ischaemia, we showed time-dependent loss of RGCs after 90 min of ischaemia after 1 week or 2 weeks.

Angiotensin II Type I Receptor Blockade Is Associated with Decreased Cutaneous Scar Formation in a Rat Model.

BACKGROUND: Angiotensin II engagement of angiotensin II type 1 receptor (AT1R) is implicated in fibrogenesis, with AT1R blockers used clinically to attenuate cardiac and renal fibrosis. The authors tested the hypothesis that the AT1R blocker losartan could reduce postsurgical cutaneous scarring in rats. METHODS: Human dermal fibroblasts were treated with losartan and assessed for viability, contractile activity, migration, and profibrotic gene transcription by means of calcein, collagen gel, scratch, and quantitative reverse transcriptase polymerase chain reaction assays, respectively. Monocyte migration and adhesion to losartan-treated and control fibroblasts were examined. Losartan effects in vivo were assessed using a mechanical distraction hypertrophic scar model. Three days after incisions were made and closed on their backs, rats were assigned randomly to receive drinking water with or without losartan (1 mg/kg per day; n = 6 per group). Distraction devices were applied and activated up to day 14. On day 28, scars underwent cross-sectional area and elevation index analyses, and α-SMA (alpha-smooth muscle actin) and CD68 (monocyte/macrophage marker) immunostaining. RESULTS: Losartan-treated human dermal fibroblasts displayed decreased contractile activity, migration, and gene expression of transforming growth factor-ß1, collagen I, and monocyte chemoattractant protein-1 relative to controls (p < 0.05). Monocyte migration and adhesion to losartan-treated fibroblasts were reduced (p < 0.01). Compared to controls, scars from losartan-treated rats demonstrated decreased cross-sectional area (19.4 ± 3.1 mm versus 45.0 ± 5.2 mm; p = 0.002), elevation index (1.5 ± 0.1 versus 2.6 ± 0.3; p = 0.003), and α-SMA and CD68 immunostaining (p < 0.001). CONCLUSIONS: Losartan decreases myofibroblast activity and reduces monocyte trafficking to cutaneous scar. These findings support losartan as a potential novel therapy for the prevention of hypertrophic scars.

Promotion of Primary Murine Breast Cancer Growth and Metastasis by Adipose-Derived Stem Cells Is Reduced in the Presence of Autologous Fat Graft.

BACKGROUND: Cell-assisted lipotransfer involves enrichment of autologous fat with supraphysiologic numbers of adipose-derived stem cells to improve graft take. Adipose-derived stem cells have been shown to promote cancer progression, raising concerns over the safety of adipose-derived stem cells and cell-assisted lipotransfer in postoncologic breast reconstruction. The authors compared the effect of adipose-derived stem cells alone, cell-assisted lipotransfer, and conventional fat grafting on breast cancer growth and metastasis. METHODS: Proliferation and migration of murine 4T1 breast cancer cells cultured in control medium or mouse adipose-derived stem cell- or fat graft-conditioned medium were assessed by flow cytometry and scratch assay, respectively. Transcription levels of arginase-1, transforming growth factor-ß, and vascular endothelial growth factor were assessed in adipose-derived stem cells and fat graft by quantitative reverse transcription polymerase chain reaction. An orthotopic mouse tumor model was used to evaluate breast cancer progression and metastasis. 4T1 cells were injected into the mammary pad of female BALB/c mice. Six days later, tumors were injected with saline, adipose-derived stem cells, fat graft, or cell-assisted lipotransfer (n = 7 per group). Two weeks later, primary tumors were examined by immunohistochemistry and lung metastasis was quantified. RESULTS: Adipose-derived stem cell-conditioned medium increased cancer cell proliferation (p = 0.03); migration (p < 0.01); and transcription of arginase-1, transforming growth factor-ß, and vascular endothelial growth factor compared to fat graft-conditioned or control medium (p < 0.02). Tumor-site injection with adipose-derived stem cells alone led to increased primary tumor growth and lung metastasis compared to control, fat graft, or cell-assisted lipotransfer groups (p < 0.05). Adipose-derived stem cell injection increased CD31 vascular density in tumors (p < 0.01). CONCLUSION: Adipose-derived stem cells alone, but not conventional fat graft or cell-assisted lipotransfer, promote breast cancer cell proliferation and invasiveness in vitro and in vivo.

Effect of Compound 21, a Selective Angiotensin II Type 2 Receptor Agonist, in a Murine Xenograft Model of Dupuytren Disease.

BACKGROUND: Although surgical excision and intralesional collagenase injection are mainstays in Dupuytren disease treatment, no effective medical therapy exists for recurrent disease. Compound 21, a selective agonist of the angiotensin II type 2 receptor, has been shown to protect against fibrosis in models of myocardial infarction and stroke. The authors investigated the potential use of compound 21 in the treatment of Dupuytren disease. METHODS: Human dermal fibroblasts were treated in vitro with compound 21 and assessed for viability using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, migration by means of scratch assay, and profibrotic gene transcription by means of quantitative reverse transcription polymerase chain reaction. Compound 21 effects in vivo were assessed using a xenograft model. Dupuytren disease cord specimens from patients undergoing open partial fasciectomy were divided into two segments. Segments were implanted under the dorsal skin of nude mouse pairs. Beginning on day 5, one mouse from each pair received daily intraperitoneal injections of compound 21 (10 µg/kg/day), and the other received vehicle. On day 10, segments were explanted and submitted for immunohistochemistry. RESULTS: Human dermal fibroblasts treated with compound 21 displayed decreased migration and decreased gene expression of connective tissue growth factor, fibroblast specific protein-1, transforming growth factor-ß1, Smad3, and Smad4. Dupuytren disease segments from compound 21-treated mice demonstrated significantly reduced alpha-smooth muscle actin and Ki67 staining, with increased density of CD31 staining vessels. CONCLUSIONS: Compound 21 significantly decreases expression of profibrotic genes and decreases myofibroblast proliferation as indicated by reduced Ki67 and alpha-smooth muscle actin expression. These findings support compound 21 as a potential novel treatment modality for Dupuytren disease.

Semiquantitative optic nerve grading scheme for determining axonal loss in experimental optic neuropathy.

PURPOSE: To describe and evaluate a semiquantitative optic nerve grading scheme for assessing axonal loss in endothelin (ET)-1-induced chronic optic neuropathy. METHODS: Optic nerve cross-sections from both eyes of 39 Brown Norway rats unilaterally treated with various concentrations of ET-1 or physiological saline solution via a surgically implanted osmotic minipump were processed for light and transmission electron microscopy (TEM). The optic nerve damage grade, between 0 (no damage) and 10 (total damage), was based on the number of zones of approximately equal damage and the mean percentage of damage within each zone. Grading was performed under light microscopy by three observers and compared with axonal survival determined with TEM using two quantification methods: the sampling method, in which approximately 10% of the section was counted, and the full-count method, in which the whole section was counted (n = 12). Axonal survival was expressed as a ratio of axon counts in the experimental to control eye. Before these comparisons, the inter- and intraobserver agreement rates were determined in another group of 85 and 12 ET-1-treated animals, respectively. RESULTS: The interobserver kappa was 0.66 (95% confidence interval [CI]: 0.58-0.74) for all eyes and 0.55 (95% CI: 0.43-0.67) for the experimental eyes only. The intraobserver kappa was 0.80, 0.81, and 0.80 for all 24 eyes and 0.60, 0.64, and 0.71 for experimental eyes only. The correlation between damage grade in the experimental eye and axonal survival using the TEM sampling method (Spearman's rho = -0.677 for all animals and -0.827 for the subset of animals with full counts only) was lower than that with the full-count method (Spearman's rho = -0.926). When axonal survival was less than 0.7, the sampling method always underestimated the extent of damage. CONCLUSIONS: The grading scheme had good inter- and intraobserver agreement, and high correlation with the TEM methods. It is a practical and time-saving method, requiring less than 1 minute per nerve and is an alternative to sampling methods that can yield significant errors.

Polysialylated Neural Cell Adhesion Molecule Protects Against Light-Induced Retinal Degeneration.

PURPOSE: We previously demonstrated that neural cell adhesion molecule (NCAM) plays an important role in supporting the survival of injured retinal ganglion cells. In the current study, we used light-induced retinal degeneration (LIRD) as a model to investigate whether NCAM plays a functional role in neuroprotection and whether NCAM influences p75NTR signaling in modulating retinal cell survival. METHODS: Retinas from wild-type (WT) and NCAM deficient (-/-) mice were tested by electroretinogram before and after LIRD, and changes in the protein expressions of NCAM, polysialic acid (PSA)-NCAM, p75NTR, and active caspase 3 were measured by immunoblot from 0 to 4 days after light induction. The effects of NCAM and PSA-NCAM on p75NTR were examined by intraocular injections of the p75NTR function-blocking antibody and/or the removal of PSA with endoneuraminidase-N prior to LIRD. RESULTS: In WT mice, low levels of active caspase 3 activation were detected on the first day, followed by increases up to 4 days after LIRD. Conversely, in NCAM-/- mice, higher cleaved caspase 3 levels along with rapid reductions in electroretinogram amplitudes were found earlier at day 1, followed by reduced levels by day 4. The removal of PSA prior to LIRD induced earlier onset of retinal cell death, an effect delayed by the coadministration of endoneuraminidase-N and the p75NTR function-blocking antibody antiserum. CONCLUSIONS: These results indicate that NCAM protects WT retinas from LIRD; furthermore, the protective effect of NCAM is, at least in part, attributed to its effects on p75NTR.

Effect of NCAM on aged-related deterioration in vision.

The neural cell adhesion molecule (NCAM) is involved in developmental processes and age-associated cognitive decline; however, little is known concerning the effects of NCAM in the visual system during aging. Using anatomical, electrophysiological, and behavioral assays, we analyzed age-related changes in visual function of NCAM deficient (-/-) and wild-type mice. Anatomical analyses indicated that aging NCAM -/- mice had fewer retinal ganglion cells, thinner retinas, and fewer photoreceptor cell layers than age-matched controls. Electroretinogram testing of retinal function in young adult NCAM -/- mice showed a 2-fold increase in a- and b-wave amplitude compared with wild-type mice, but the retinal activity dropped dramatically to control levels when the animals reached 10 months. In behavioral tasks, NCAM -/- mice had no visual pattern discrimination ability and showed premature loss of vision as they aged. Together, these findings demonstrate that NCAM plays significant roles in the adult visual system in establishing normal retinal anatomy, physiology and function, and in maintaining vision during aging.

Effect of intraocular pressure on optic disc topography, electroretinography, and axonal loss in a chronic pressure-induced rat model of optic nerve damage.

PURPOSE: To characterize the effect of intraocular pressure (IOP) on optic disc topography, retinal function, and axonal survival in a model of IOP-induced optic nerve damage in rat. METHODS: Hypertonic (1.75 M) saline was injected into an episcleral vein of one eye of 49 Brown Norway rats, with the fellow untreated eye serving as the control. During the 1 to 3 months of follow-up, IOP was measured twice weekly in conscious animals with a handheld tonometer, and changes in disc topography and retinal function were monitored with scanning laser tomography and electroretinography (ERG), respectively. Peak IOP elevation in the experimental eye compared with the fellow control eye (peak deltaIOP), integral of IOP elevation over time (deltaIOP integral), and days of IOP elevation were calculated. Axon counts were obtained from electron micrographs of the sectioned optic nerves. RESULTS: Progressive cupping was found in 9 (56.3%) of 16 eyes with peak deltaIOP of more than 15 mm Hg and in none of 21 eyes with peak deltaIOP less than 15 mm Hg. A strong correlation between deltaIOP integral and progressive cupping was also found, but not with days of IOP elevation. ERG abnormalities (limited to the b-wave) were found in 11 (64.7%) of 17 eyes with peak deltaIOP of more than 15 mm Hg and in 2 (8.7%) of 23 eyes with peak deltaIOP of less than 15 mm Hg. Neither of the other IOP parameters was predictive of ERG damage. The proportion of surviving axons was negatively correlated to both deltaIOP and deltaIOP integral (P

Effects of cold-induced vasospasm in glaucoma: the role of endothelin-1.

PURPOSE: Vasospasm has been associated with glaucoma, but its mechanisms have not been elucidated. The present study was designed to evaluate the role of endothelin (ET)-1, a potent endogenous vasoconstrictor, in the genesis of vasospasm in glaucoma. METHODS: Our sample contained patients with open-angle glaucoma (n = 43) and subjects with normal nonglaucomatous eyes and without acral vasospasm (n = 27). After the eligibility visit, all subjects underwent a provocative cooling test, consisting of wearing for 30 minutes a head-vest cooling garment containing coolant fluid. Blood was collected before and after cooling, and plasma ET-1 was determined by immunoassay. In addition, visual fields and retinal blood flow, measured with a confocal scanning laser and Doppler flowmeter, were measured before and after cooling. Peripheral finger flow, skin temperature, and blood pressure were monitored during the experiment. A recovery visit was performed within 1 month, when visual field and retinal blood flow measurements were repeated. RESULTS: Baseline plasma ET-1 levels were similar between patients with glaucoma and control subjects (mean +/- SD: 2.81 +/- 1.29 and 2.56 +/- 1.36 pg/mL, respectively, P = 0.465). Patients with glaucoma, however, had a significant increase in plasma ET-1 after cooling (mean +/- SD increase of 34% +/- 52%, P = 0.001), not observed in control subjects (mean +/- SD increase of 7% +/- 43%, P = 0.750). No significant change in visual fields or retinal blood flow was observed after cooling in either group. Patients with glaucoma who had evidence of acral vasospasm, however, were more likely to show deterioration in visual fields after cooling than patients without acral vasospasm (P = 0.007). CONCLUSIONS: Patients with glaucoma have an abnormal increase in plasma ET-1 after the body cools. It is possible that at least in some patients, increased levels of ET-1 in response to vasospastic stimuli may be involved in the pathogenesis of glaucomatous damage.

Model of endothelin-1-induced chronic optic neuropathy in rat.

PURPOSE: To describe a model of chronic endothelin (ET)-1 administration to the optic nerve and evaluate its effect on retinal ganglion cell (RGC) and axon survival in rat. METHODS: Osmotic minipumps were surgically implanted in one eye of 113 Brown Norway rats to deliver 0.05, 0.10, 0.20, or 0.40 microg ET-1 per day (3.3, 6.7, 13.4, and 26.8 microM, respectively), or balanced salt solution (BSS) to the immediate retrobulbar optic nerve; the fellow untreated eye served as the control. Before pump implantation, RGCs were retrogradely labeled with fluorochrome. Animals were killed at 21, 42, or 84 days. RGC survival was expressed as the ratio of RGC counts in experimental versus control eyes in wholemounted retinas, whereas axon survival was expressed similarly from electron micrographs of the optic nerves. Serial optic disc changes were evaluated using scanning laser tomography. The effect of ET-1 (3 microL topical application of 10(-5) M) on blood flow in the surgically exposed optic nerve was measured using laser Doppler flowmetry in a separate group of five animals. RESULTS: ET-1 led to a mean reduction in optic nerve blood flow of 68%. There were no significant differences in RGC survival among the four ET-1 doses used in this study. Pooled across all ET-1 doses, RGC survival decreased incrementally at 21, 42, and 84 days (P < 0.001; mean +/- SD, 0.77 +/- 0.25, 0.60 +/- 0.27, and 0.50 +/- 0.26, respectively) and was statistically significantly lower at each time point than in the BSS-treated animals. The axon survival data also showed a similar time-dependent loss. Only one of 21 animals showed significantly increased disc cupping, and there was no relationship between RGC survival and change in cupping. CONCLUSIONS. Chronic administration of ET-1 to the rat optic nerve results in a time-dependent loss of RGCs and their axons without apparent change in optic disc topography.

Timolol concentrations in rat ocular tissues and plasma after topical and intraperitoneal dosing.

PURPOSE: Topical beta-blockers, such as timolol, have been used extensively in the medical treatment of glaucoma to lower intraocular pressure (IOP). Recently, these drugs have been shown to have effects on the retinal and optic nerve circulation as well as potential neuroprotective properties. In the current study, the concentration of timolol attained in the cornea, iris-ciliary body, retina, vitreous, and plasma was measured after topical or intraperitoneal administration in rats to determine the relative contributions of each route to intraocular timolol concentrations. MATERIALS AND METHODS: One group of rats received one drop of commercially available 0.5% timolol in the right eye and two drops in the left eye for 3 to 12 days. Another group of rats received one drop of 0.5% timolol in one eye only and concentrations were studied in the ocular tissues at 15, 30, 60, 120, and 240 minutes after instillation. The final group of rats received a single intraperitoneal injection of timolol ranging in concentration from 5 to 75 mg/kg after which tissue and plasma concentrations were measured 30 minutes after injection. All tissue and plasma concentrations were measured by high performance liquid chromatography. RESULTS: Rats that received topical timolol daily for 3 to 12 consecutive days accumulated timolol concentrations of 2.3 to 4.4 microg/g in cornea, 198 to 326 microg/g in iris, 0.05 to 0.11 microg/ml in vitreous, and 0.17 to 0.77 microg/g in retina. In rats that received a single drop of timolol in one eye, the tissue concentrations were higher in the treated eye than in the untreated eye in all cases except for vitreous. In these experiments, timolol levels in plasma were either low or not detectable. Increasing timolol doses administered intraperitoneally resulted in corresponding increased tissue and plasma concentrations. CONCLUSIONS: Absorption of drug into the systemic circulation plays a significant role in delivering timolol to the retina and vitreous in addition to a local ocular route. A clear dose-response relationship exists in all ocular tissues studied after an intraperitoneal dose of timolol. High doses of timolol were required to achieve measurable concentrations of drug in the ocular tissues via our high performance liquid chromatography assay suggesting that a significant hepatic first-pass effect may be involved after an intraperitoneal injection of timolol.