Robert H. Rosa

Action at a Distance: Systemically Administered Adult Stem/Progenitor Cells (MSCs) Reduce Inflammatory Damage to the Cornea Without Engraftment and Primarily by Secretion of TNF‐α Stimulated Gene/Protein 6

Previous reports demonstrated that the deleterious effects of chemical injury to the cornea were ameliorated by local or systemic administration of adult stem/progenitor cells from bone marrow referred to as mesenchymal stem or stromal cells (MSCs). However, the mechanisms for the beneficial effects of MSCs on the injured cornea were not clarified. Herein, we demonstrated that human MSCs (hMSCs) were effective in reducing corneal opacity and inflammation without engraftment after either intraperitoneal (i.p.) or intravenous (i.v.) administration following chemical injury to the rat cornea. A quantitative assay for human mRNA for glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) demonstrated that less than 10 hMSCs were present in the corneas of rats 1‐day and 3 days after i.v. or i.p. administration of 1 × 107 hMSCs. In vitro experiments using a transwell coculture system demonstrated that chemical injury to corneal epithelial cells activated hMSCs to secrete the multipotent anti‐inflammatory protein TNF‐α stimulated gene/protein 6 (TSG‐6). In vivo, the effects of i.v. injection of hMSCs were largely abrogated by knockdown of TSG‐6. Also, the effects of hMSCs were essentially duplicated by either i.v. or topical administration of TSG‐6. Therefore, the results demonstrated that systemically administered hMSCs reduce inflammatory damage to the cornea without engraftment and primarily by secretion of the anti‐inflammatory protein TSG‐6 in response to injury signals from the cornea. STEM CELLS 2011;29:1572–1579

Anti-inflammatory protein TSG-6 reduces inflammatory damage to the cornea following chemical and mechanical injury

Previous reports demonstrated that adult stem/progenitor cells from bone marrow (multipotent mesenchymal stem cells; MSCs) can repair injured tissues with little evidence of engraftment or differentiation. In exploring this phenomenon, our group has recently discovered that the therapeutic benefits of MSCs are in part explained by the cells being activated by signals from injured tissues to express an anti-inflammatory protein TNF-α–stimulated gene/protein 6 (TSG-6). Therefore, we elected to test the hypothesis that TSG-6 would have therapeutic effects in inflammatory but noninfectious diseases of the corneal surface. We produced a chemical and mechanical injury of the cornea in rats by brief application of 100% ethanol followed by mechanical debridement of corneal and limbal epithelium. Recombinant human TSG-6 or PBS solution was then injected into the anterior chamber of the eye. TSG-6 markedly decreased corneal opacity, neovascularization, and neutrophil infiltration. The levels of proinflammatory cytokines, chemokines, and matrix metalloproteinases were also decreased. The data indicated that TSG-6, a therapeutic protein produced by MSCs in response to injury signals, can protect the corneal surface from the excessive inflammatory response following injury.

Divergent Roles of Nitric Oxide and Rho Kinase in Vasomotor Regulation of Human Retinal Arterioles

PURPOSEnAlthough the arteriolar segment contributes to flow regulation, there is sparse information at the single microvessel level on how vasomotor function is regulated in the human retina. The authors have previously reported vasoreactivity and its underlying mechanisms in isolated porcine retinal arterioles. Herein, they studied human retinal arterioles for comparison.nnnMETHODSnRetinal tissues were obtained from seven patients undergoing enucleation. Human and porcine retinal arterioles were isolated and pressurized to 55 cm H(2)O luminal pressure for vasoreactivity study using videomicroscopic techniques.nnnRESULTSnIsolated human and porcine retinal arterioles developed myogenic tone and dilated dose dependently to bradykinin, adenosine, and sodium nitroprusside. Stepwise increases in luminal flow produced graded dilation with approximately 60% dilation at the highest flow tested. Nitric oxide (NO) synthase inhibitor L-NAME nearly abolished dilations to bradykinin and flow and attenuated the adenosine-induced dilation without altering the response to nitroprusside. Endothelin-1 caused dose-dependent constriction. Rho kinase (ROCK) inhibitor H-1152 blocked both myogenic tone and endothelin-1-induced constriction. Responses of retinal arterioles to all agonists and increased flow were similar between pigs and humans.nnnCONCLUSIONSnIsolated human retinal arterioles dilate to bradykinin and increased flow in an NO-dependent manner. NO contributes, in part, to adenosine-induced vasodilation. Conversely, ROCK activation mediates myogenic tone and endothelin-1-induced vasoconstriction. Similarities in these vasoactive responses and the underlying mechanisms between human and porcine retinal arterioles support the latter as a viable experimental model of the human retinal microcirculation.

Stanniocalcin-1 Rescued Photoreceptor Degeneration in Two Rat Models of Inherited Retinal Degeneration

Oxidative stress and photoreceptor apoptosis are prominent features of many forms of retinal degeneration (RD) for which there are currently no effective therapies. We previously observed that mesenchymal stem/stromal cells reduce apoptosis by being activated to secrete stanniocalcin-1 (STC-1), a multifunctional protein that reduces oxidative stress by upregulating mitochondrial uncoupling protein-2 (UCP-2). Therefore, we tested the hypothesis that intravitreal injection of STC-1 can rescue photoreceptors. We first tested STC-1 in the rhodopsin transgenic rat characterized by rapid photoreceptor loss. Intravitreal STC-1 decreased the loss of photoreceptor nuclei and transcripts and resulted in measurable retinal function when none is otherwise present in this rapid degeneration. We then tested STC-1 in the Royal College of Surgeons (RCS) rat characterized by a slower photoreceptor degeneration. Intravitreal STC-1 reduced the number of pyknotic nuclei in photoreceptors, delayed the loss of photoreceptor transcripts, and improved function of rod photoreceptors. Additionally, STC-1 upregulated UCP-2 and decreased levels of two protein adducts generated by reactive oxygen species (ROS). Microarrays from the two models demonstrated that STC-1 upregulated expression of a similar profile of genes for retinal development and function. The results suggested that intravitreal STC-1 is a promising therapy for various forms of RD including retinitis pigmentosa and atrophic age-related macular degeneration (AMD).Oxidative stress and photoreceptor apoptosis are prominent features of many forms of retinal degeneration (RD) for which there are currently no effective therapies. We previously observed that mesenchymal stem/stromal cells reduce apoptosis by being activated to secrete stanniocalcin-1 (STC-1), a multifunctional protein that reduces oxidative stress by upregulating mitochondrial uncoupling protein-2 (UCP-2). Therefore, we tested the hypothesis that intravitreal injection of STC-1 can rescue photoreceptors. We first tested STC-1 in the rhodopsin transgenic rat characterized by rapid photoreceptor loss. Intravitreal STC-1 decreased the loss of photoreceptor nuclei and transcripts and resulted in measurable retinal function when none is otherwise present in this rapid degeneration. We then tested STC-1 in the Royal College of Surgeons (RCS) rat characterized by a slower photoreceptor degeneration. Intravitreal STC-1 reduced the number of pyknotic nuclei in photoreceptors, delayed the loss of photoreceptor transcripts, and improved function of rod photoreceptors. Additionally, STC-1 upregulated UCP-2 and decreased levels of two protein adducts generated by reactive oxygen species (ROS). Microarrays from the two models demonstrated that STC-1 upregulated expression of a similar profile of genes for retinal development and function. The results suggested that intravitreal STC-1 is a promising therapy for various forms of RD including retinitis pigmentosa and atrophic age-related macular degeneration (AMD).

Sildenafil (Viagra) Evokes Retinal Arteriolar Dilation: Dual Pathways via NOS Activation and Phosphodiesterase Inhibition

PURPOSEnSildenafil (Viagra; Pfizer, New York, NY), a selective phosphodiesterase type-5 (PDE5) inhibitor, is widely used to treat impotence by improving penile blood flow via elevation of cGMP. However, its effect on ocular circulation is controversial and whether retinal arterioles are responsive to this drug remains unclear. In this study, the direct reaction of retinal arterioles to sildenafil was examined and the signaling pathway underlying this vasomotor activity was probed.nnnMETHODSnRetinal arterioles from porcine eyes were isolated, cannulated, and pressurized without flow. Diameter changes in response to sildenafil were recorded using videomicroscopic techniques.nnnRESULTSnRetinal arterioles (67 +/- 2 microm) dilated dose dependently to sildenafil (1 ng/mL to 1 microg/mL). This dilation was inhibited by the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), the guanylyl cyclase inhibitor 1H- [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), the extracellular signal-regulated kinase (ERK) pathway inhibitor PD98059, the nonselective potassium channel blocker tetraethylammonium (TEA), and the selective adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channel blocker glibenclamide. The vasodilation elicited by the NO donor S-nitroso-N-acetylpenicillamine (SNAP) was inhibited by ODQ and TEA but was insensitive to PD98059. In the presence of L-NAME, the addition of SNAP (1 microM) produced modest vasodilation and the inhibited sildenafil response was subsequently restored. The restored dilation was insensitive to PD98059 but was blocked by TEA.nnnCONCLUSIONSnActivation of NO synthase, through ERK signaling, leading to NO production and subsequent guanylyl cyclase activation and K(ATP) channel opening is the major vasodilatory pathway for sildenafil in retinal arterioles. Moreover, the elevated cGMP, from endogenous or exogenous NO, plays a permissive role for sildenafil to exert vasodilation through inhibition of the PDE5 pathway independent of ERK signaling.

Acute Retinal Ischemia Inhibits Endothelium-Dependent Nitric Oxide–Mediated Dilation of Retinal Arterioles via Enhanced Superoxide Production

PURPOSEnBecause retinal vascular disease is associated with ischemia and increased oxidative stress, the vasodilator function of retinal arterioles was examined after retinal ischemia induced by elevated intraocular pressure (IOP). The role of superoxide anions in the development of vascular dysfunction was assessed.nnnMETHODSnIOP was increased and maintained at 80 to 90 mm Hg for 30, 60, or 90 minutes by infusing saline into the anterior chamber of a porcine eye. The fellow eye with normal IOP (10-20 mm Hg) served as control. In some pigs, superoxide dismutase mimetic TEMPOL (1 mM) or vehicle (saline) was injected intravitreally before IOP elevation. After enucleation, retinal arterioles were isolated and pressurized without flow for functional analysis by recording diameter changes using videomicroscopic techniques. Dihydroethidium (DHE) was used to detect superoxide production in isolated retinal arterioles.nnnRESULTSnIsolated retinal arterioles developed stable basal tone and the vasodilations to endothelium-dependent nitric oxide (NO)-mediated agonists bradykinin and L-lactate were significantly reduced only by 90 minutes of ischemia. However, vasodilation to endothelium-independent NO donor sodium nitroprusside was unaffected after all time periods of ischemia. DHE staining showed that 90 minutes of ischemia significantly increased superoxide levels in retinal arterioles. Intravitreal injection of membrane-permeable radical scavenger but not vehicle before ischemia prevented elevation of vascular superoxide and preserved bradykinin-induced dilation.nnnCONCLUSIONSnEndothelium-dependent NO-mediated dilation of retinal arterioles is impaired by 90 minutes of ischemia induced by elevated IOP. The inhibitory effect appears to be mediated by the alteration of NO signaling via vascular superoxide.

Alterations of Ocular Hemodynamics Impair Ophthalmic Vascular and Neuroretinal Function

Hypertension is associated with numerous diseases, but its direct impact on the ocular circulation and neuroretinal function remains unclear. Herein, mouse eyes were challenged with different levels of hemodynamic insult via transverse aortic coarctation, which increased blood pressure and flow velocity by 50% and 40%, respectively, in the right common carotid artery, and reduced those parameters by 30% and 40%, respectively, in the left common carotid artery. Blood velocity in the right central retinal artery gradually increased up to 40% at 4 weeks of transverse aortic coarctation, and the velocity in the left central retinal artery gradually decreased by 20%. The fundus and retinal architecture were unaltered by hemodynamic changes. Endothelium-dependent vasodilations to acetylcholine and adenosine were reduced only in right (hypertensive) ophthalmic arteries. Increased cellularity in the nerve fiber/ganglion cell layers, enhanced glial fibrillary acidic protein expression, and elevated superoxide level were found only in hypertensive retinas. The electroretinogram showed decreased scotopic b-waves in the hypertensive eyes and decreased scotopic oscillatory potentials in both hypertensive and hypotensive eyes. In conclusion, hypertension sustained for 4 weeks causes ophthalmic vascular dysfunction, retinal glial cell activation, oxidative stress, and neuroretinal impairment. Although ophthalmic vasoregulation is insensitive to hypotensive insult, the ocular hypoperfusion causes neuroretinal dysfunction.