MARCONY RODRIGUES DE SANTHIAGO
Projetos de Pesquisa
Unidades Organizacionais
LIM/33 - Laboratório de Oftalmologia, Hospital das Clínicas, Faculdade de Medicina
9 resultados
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Agora exibindo 1 - 9 de 9
- Topical Losartan Decreases Myofibroblast Generation But Not Corneal Opacity After Surface Blast-Simulating Irregular PTK in Rabbits(2023) SAMPAIO, Lycia Pedral; VILLABONA-MARTINEZ, Valeria; SHIJU, Thomas Michael; SANTHIAGO, Marcony R.; WILSON, Steven E.Purpose: To evaluate the efficacy of topical losartan after blast injury-simulating irregular phototherapeutic keratectomy (PTK) in rabbits.Methods: Twelve NZW rabbits underwent 100 pulse 6.5 mm diameter PTK over a metal screen to generate severe surface irregularity and inhibit epithelial basement membrane regeneration. Corneas were treated with 0.8 mg/mL losartan in balanced salt solution (BSS) or BSS 50 mu L six times per day for six weeks after PTK. All corneas had slit lamp photography, with and without 1% fluorescein at two, four, and six weeks after PTK, and were analyzed using immunohistochemistry for the myofibroblast marker alpha-smooth muscle actin (alpha-SMA), keratocyte marker keratocan, mesenchymal cell marker vimentin, transforming growth factor (TGF)-beta 1, and collagen type IV.Results: Topical 0.8 mg/mL losartan six times a day significantly decreased anterior stromal alpha-SMA intensity units compared to BSS at six weeks after anterior stromal irregularity-inducing screened PTK (P = 0.009). Central corneal opacity, however, was not significantly different between the two groups. Keratocan, vimentin, TGF-beta 1, or collagen type IV levels in the anterior stroma were not significantly different between the two groups.Conclusions: Topical losartan effectively decreased myofibroblast generation after surface blast simulation irregular PTK. However, these results suggest initial maskingsmoothing PTK, along with adjuvant topical losartan therapy, may be needed to decrease corneal stromal opacity after traumatic injuries that produce severe surface irregularity.Translational Relevance: Topical losartan decreased scar-producing stromal myofibroblasts after irregular PTK over a metal screen but early smoothing of irregularity would also likely be needed to significantly decrease corneal opacity.
conferenceObject Cell biology of spontaneous persistent epithelial defects after photorefractive keratectomy in rabbits(2023) WILSON, Steven; SAMPAIO, Lycia; VILLABONA-MARTINEZ, Valeria; MICHAEL, Shiju; HILGERT, Guilherme; SANTHIAGO, Marcony- Corneal stromal localization of TGF beta isoforms in spontaneous persistent epithelial defects after PRK in rabbits(2024) VILLABONA-MARTINEZ, Valeria; DUTRA, Barbara A. L.; SAMPAIO, Lycia P.; SANTHIAGO, Marcony R.; WILSON, Steven E.The purpose of this study was to evaluate transforming growth factor beta (TGFI3) isoform localization in rabbit corneas with spontaneous persistent epithelial defects (PEDs) after photorefractive keratectomy (PRK). Four cryofixed corneas from a previously reported series of PEDs in rabbits that had PRK were evaluated with triplex immunohistochemistry (IHC) for TGFI33, myofibroblast marker alpha-smooth muscle actin (alpha-SMA) and mesenchymal marker vimentin. One cornea had sufficient remaining tissue for triplex IHC for TGFI31, TGFI32, or TGFI33 (each with alpha-SMA and vimentin) using isoform-specific antibodies. All three TGFI3 isoforms were detected in the subepithelial stroma at and surrounding the PED. Some of each TGFI3 isoform co-localized with alpha-SMA of myofibroblasts, which could be TGFI3 isoform autocrine production by myofibroblasts or TGFI3-1, -2, and -3 binding to these myofibroblasts.
- Topical losartan inhibits corneal scarring fibrosis and collagen type IV deposition after Descemet's membrane-endothelial excision in rabbits (vol 216, 108940, 2022)(2022) SAMPAIO, L. P.; HILGERT, G. S. L.; SHIJU, T. M.; SANTHIAGO, M. R.; WILSON, S. E.
- Descemet's membrane injury and regeneration, and posterior corneal fibrosis, in rabbits(2021) SAMPAIO, Lycia Pedral; SHIJU, Thomas Michael; HILGERT, Guilherme S. L.; OLIVEIRA, Rodrigo Carlos de; DEDREU, JodiRae; MENKO, A. Sue; SANTHIAGO, Marcony R.; WILSON, Steven E.The purpose of this investigation was to study Descemet's membrane and corneal endothelial regeneration, myofibroblast generation and disappearance, and TGF beta-1 localization after Descemet's membraneendothelial excision (Descemetorhexis) in rabbits. Thirty-six rabbits had 8 mm Descemetorhexis and standardized slit lamp photos at 1, 2 and 4 days, 1, 2 and 4 weeks, and 2, 4 and 6 months, as well as multiplex IHC for stromal cell markers keratocan, vimentin, and alpha-smooth muscle actin (SMA); basement membrane (BM) components perlecan, nidogen-1, laminin alpha-5, and collagen type IV; and corneal endothelial marker Na,KATPase beta 1, and TGF beta-1, with ImageJ quantitation. Stromal transparency increased from the periphery beginning at two months after injury and progressed into the central cornea by six months. At six months, central transparency was primarily limited by persistent mid-stromal neovascularization. Stromal myofibroblast zone thickness in the posterior stroma peaked at one month after injury, and then progressively decreased until to six months when few myofibroblasts remained. The regeneration of a laminin alpha-5 and nidogen-1 Descemet's membrane ""railroad track"" structure was accompanied by corneal endothelial closure and stromal cell production of BM components in corneas from four to six months after injury. TGF beta-1 deposition at the posterior corneal surface from the aqueous humor peaked at one day after Descemetorhexis and diminished even before regeneration of the endothelium and Descemet's membrane. This decrease was associated with collagen type IV protein production by corneal fibroblasts, and possibly myofibroblasts, in the posterior stroma. Descemet's membrane and the corneal endothelium regenerated in the rabbit cornea by six months after eight mm Descemetorhexis. Real-time quantitative RT-PCR experiments in vitro with marker-verified rabbit corneal cells found that 5 ng/ml or 10 ng/ml TGF beta-1 upregulated col4a1 or col4a2 mRNA expression after 6 h or 12 h of exposure in corneal fibroblasts, but not in myofibroblasts. Stromal cells produced large amounts of collagen type IV that likely decreased TGF beta-1 penetration into the stroma and facilitated the resolution of myofibroblastgenerated fibrosis.
- Topical losartan inhibits corneal scarring fibrosis and collagen type IV deposition after Descemet's membrane-endothelial excision in rabbits(2022) SAMPAIO, Lycia Pedral; HILGERT, Guilherme S. L.; SHIJU, Thomas Michael; MUEILLO, Sofia E.; SANTHIAGO, Marcony R.; WILSON, Steven E.The purpose of this study was to examine the effect of topical and/or oral angiotensin converting enzyme II inhibitor and TGF-beta signaling blocker losartan on corneal stromal fibrosis that developed in rabbit corneas after Descemetorhexis removal of central Descemet's membrane and corneal endothelium. Twenty-eight New Zealand white rabbits were included and either had 8 mm central Descemetorhexis or sham control surgery without Descemetorhexis in one eye. Groups of 4 eyes without Descemetorhexis were treated for one month with no medications, topical losartan or oral losartan. Groups of 4 eyes with Descemetorhexis were treated with topical and oral vehicle, topical losartan, oral losartan, or both topical losartan and oral losartan for one month. Standardized slit lamp photos were obtained with central opacity intensity measured with ImageJ. The posterior fibrotic zone of corneas was measured on immunohistochemistry for alpha-smooth muscle actin (SMA) and keratocan using QuPath analysis. Collagen type IV expression in the posterior cornea was quantitated with ImageJ and duplex immunohistochemistry for collagen type IV and TGF beta-1. After Descemetorhexis, topical, but not oral, losartan decreased the intensity of central stromal opacity, reduced peripheral corneal scarring, and decreased alpha-smooth muscle actin myofibroblast fibrosis area compared to corneas that had Descemetorhexis and treatment with vehicles alone. Topical losartan decreased posterior stromal cellular, non-Descemet's membrane, collagen type IV production, that is likely stimulated by TGF beta as part of a negative regulatory feedback mechanism, compared to vehicle treatment at one month after Descemetorhexis. Topical losartan is likely to be effective in reducing corneal scarring fibrosis produced by traumatic injury, microbial infection, and some corneal diseases and surgeries.
- Topical Losartan and Corticosteroid Additively Inhibit Corneal Stromal Myofibroblast Generation and Scarring Fibrosis After Alkali Burn Injury(2022) SAMPAIO, Lycia Pedral; HILGERT, Guilherme S. L.; SHIJU, Thomas Michael; SANTHIAGO, Marcony R.; WILSON, Steven E.Purpose: To evaluate the efficacy of losartan and prednisolone acetate in inhibiting corneal scarring fibrosis after alkali burn injury in rabbits. Methods: Sixteen New Zealand White rabbits were included. Alkali injuries were produced using 1N sodium hydroxide on a 5-mm diameterWhatman #1 filter paper for 1 minute. Four corneas in each group were treated six times per day for 1 month with 50 mu L of (1) 0.2 mg/mL losartan in balanced salt solution (BSS), (2) 1% prednisolone acetate, (3) combined 0.2 mg/mL losartan and 1% prednisolone acetate, or (4) BSS. Area of opacity and total opacitywere analyzed in standardized slit-lamp photoswith ImageJ. Corneas in both groupswere cryofixed in Optimal cutting temperature (OCT) compound at 1 month after surgery, and immunohistochemistry was performed for alpha-smooth muscle actin (alpha-SMA) and keratocan or transforming growth factor beta 1 and collagen type IV with ImageJ quantitation. Results: Combined topical losartan and prednisolone acetate significantly decreased slit-lamp opacity area and intensity, as well as decreased stromal myofibroblast alpha-SMA area and intensity of staining per section and confinedmyofibroblasts to only the posterior stroma with repopulation of the anterior and mid-stroma with keratocan-positive keratocytes after 1 month of treatment. Corneal fibroblasts produced collagen type IV not associated with basement membranes, and this production was decreased by topical losartan. Conclusions: Combined topical losartan and prednisolone acetate decreased myofibroblast-associated fibrosis after corneal alkali burns that produced full-thickness injury, including corneal endothelial damage. Increased dosages and duration of treatment may further decrease scarring fibrosis. Translational Relevance: Topical losartan and prednisolone acetate decrease myofibroblast-mediated scarring fibrosis after corneal injury.
- Cell Biology of Spontaneous Persistent Epithelial Defects After Photorefractive Keratectomy in Rabbits(2023) SAMPAIO, Lycia Pedral; MARTINEZ, Valeria Villabona; SHIJU, Thomas Michael; HILGERT, Guilherme S. L.; SANTHIAGO, Marcony R.; WILSON, Steven E.Purpose: To evaluate wound healing in rabbit corneas that developed a spontaneous persistent epithelial defect (PED) after photorefractive keratectomy (PRK). Methods: Forty-eight 10- to 15-week-old female New Zealand White rabbits weighing 2.5 to 3.0 kg underwent either -3 diopter (D) or -9 D PRK to generate a series of corneas to study wound healing after injury. During that series, seven corneas developed a PED detected with 1% fluorescein staining at a slit lamp that either did not have epithelial closure by 1 week after surgery or subsequently had the closed epithelium break down to form a PED 2 to 3 weeks after surgery. The corneas had slit-lamp photography, with and without 1% fluorescein, and were removed from the normal PRK series. Each PED cornea was evaluated using immunohistochemistry for the myofibroblast marker & alpha;-smooth muscle actin (& alpha;-SMA), keratocyte marker keratocan, and mesenchymal cell marker vimentin, as well as basement membrane components perlecan and collagen type IV. Results: All seven corneas that had PRK with a PED, even the two evaluated at only 1 week after PRK, had & alpha;-SMA-positive myofibroblasts populating the anterior stroma within the PED, along with comingled & alpha;-SMA-negative cells that were likely corneal fibroblasts and possibly bone marrow-derived fibrocytes. Both perlecan and collagen type IV accumulated in the anterior stroma of the epithelial defects without an epithelial basement membrane, likely produced by corneal fibroblasts to modulate transforming growth factor-& beta; entering the stroma from the tears and peripheral epithelium. Conclusions: Corneas with a PED that occurred following PRK (a procedure that produces a transient neurotropic state in the cornea) had myofibroblasts populating the superficial stroma within the epithelial defect as early as 1 week after the surgery. Translational Relevance: Pharmacologic treatments that trigger myofibroblast apoptosis, including topical losartan, could facilitate decreased scarring fibrosis in corneas with a PED.
- p Epithelial Basement Membrane Regeneration After PRK-Induced Epithelial-Stromal Injury in Rabbits: Fibrotic Versus Non-fibrotic Corneal Healing(2022) OLIVEIRA, Rodrigo Carlos de; SAMPAIO, Lycia Pedral; SHIJU, Thomas Michael; SANTHIAGO, Marcony R.; WILSON, Steven E.PURPOSE: To study epithelial basement membrane (EBM) regeneration in non-fibrotic and fibrotic corneas after photorefractive keratectomy (PRK). METHODS: Rabbits (120 total) had either epithelial scrape alone,-4.50 diopters (D) PRK,-9.00 D PRK, or no surgery. Immunohistochemistry was performed on cryofixed corneas at time points from unwounded to 8 weeks (four corneas at each time point in each group). Multiplex immunohistochemistry was performed for EBM components, including collagen type IV, laminin beta-3, laminin alpha-5, perlecan, and nidogen-1. Stromal cellular composition was studied by triplex immunohistochemistry for keratocan, vimentin, and alpha-smooth muscle actin (SMA). RESULTS: PRK-injured EBM significantly regenerated by 4 days after surgery. However, early TOE-beta-regulating perlecan incorporation into the nascent EBM declined 4 to 7 days after surgery in fibrotic corneas. Non-fibrotic corneas that had fully regenerated EBM (with all five components incorporated into the EBM) were transparent and had few SMApositive myofibroblasts in the stroma. Conversely, corneas with defective nascent EBM that lacked perlecan developed many anterior stromal myofibroblasts and fibrosis at 3 to 4 weeks after surgery and had large amounts of collagen type IV in the nascent EBM and anterior stroma. Myofibroblasts synthesized perlecan but were incompetent to incorporate the heparin sulfate proteoglycan into the nascent EBM. Corneal transparency was restored over several months even in fibrotic corneas, and this was associated with a return of EBM perlecan, myofibroblast disappearance, and reabsorption of disordered extracellular matrix. CONCLUSIONS: Defective incorporation of perlecan into the regenerating EBM by subepithelial myofibroblasts, and likely their precursor cells, underlies the development and persistence of stromal fibrosis after PRK corneal injury. [J Refract Surg. 2022;38(1):50-60.]