V. Agrawal

Effect of TGFβ and PDGF-B blockade on corneal myofibroblast development in mice

The purpose of this study was to investigate the role of transforming growth factor beta (TGFβ) and/or platelet-derived growth factor-B (PDGF-B) blockade on the differentiation of vimentin and alpha-smooth muscle actin (αSMA)-expressing myofibroblasts associated with haze in mice. Mouse corneas had haze-generating irregular PTK (phototherapeutic keratectomy) and topical treatment with the vectors. Six study groups of PTK treated corneas, with four corneas per group in each experiment, were Group 1) treated with TGFβ-KDEL vector interfering with TGFβ signaling through anomalous sorting of cytokine bound to the expressed altered receptor; Group 2) treated with PDGF-B-KDEL vector interfering with PDGF signaling through anomalous sorting of cytokine bound to the expressed altered receptor; Group 3) treated with both TGFβ-KDEL vector and PDGF-B-KDEL vector to interfere with signaling of both cytokines; Group 4) empty pGFPC1 vector; Group 5) empty pCMV vector; and Group 6) no vector treatment control. At one month after surgery, the corneas were analyzed by immunocytochemistry (IHC) for central stromal cells expressing myofibroblast markers vimentin and αSMA. The stroma of corneas treated with the TGFβ-KDEL vector alone (p < 0.05) or both the TGFβ-KDEL and PDGF-B-KDEL vectors (P < 0.05) had significantly lower density of vimentin-positive cells compared to the corresponding control group. The central stroma of corneas treated with the TGFβ-KDEL vector (p < 0.05) or the PDGF-B-KDEL vector (p < 0.05) had lower density of αSMA-positive cells compared to the corresponding control group. The density of αSMA-positive stromal cells was also significantly lower (p < 0.05) when both the TGFβ-KDEL and PDGF-B-KDEL and vectors were applied together compared to the corresponding control groups. This study provides in situ evidence that TGFβ and PDGF-B have important roles in modulating myofibroblast generation in the mouse cornea after haze-associated injury.

Monocyte development inhibitor PRM-151 decreases corneal myofibroblast generation in rabbits.

This study investigated whether PRM-151 (Promedior, Inc., Malvern, PA), a recombinant form of human pentraxin-2 (PTX-2, also referred to as serum amyloid P, hSAP), that inhibits differentiation of circulating monocytes into fibrocytes and profibrotic macrophages, could modulate generation of myofibroblasts after opacity-producing corneal injury in rabbits, and, therefore, have potential to reduce or prevent haze after PRK. Nine diopter PRK for myopia was performed with the VISX S4 IR laser. Four groups of 6 animals were treated in masked fashion: Group 1: 30 μl of topical PRM-151 (20 mg/ml) 6 times a day for 5 days; Group 2: 30 μl topical vehicle 6 times a day for 5 days; Group 3: 200 μl sub-conjunctival PRM-151 (total injection of 4 mg) immediately after surgery and every other day until day 8; Group 4: 200 μl sub-conjunctival injections of vehicle according to the same schedule as group 3. At one month after PRK, the animals were euthanized and immunohistochemistry was performed for the myofibroblast marker α-smooth muscle actin (SMA). The density of SMA+ cells/400× field in the central stroma was determined in each cornea. Myofibroblast density at one month after surgery was significantly lower (p = 0.006) after sub-conjunctival PRM-151 treatment (5.8 ± 2.8 cells/400× stromal field) compared to sub-conjunctival vehicle treatment (15.3 ± 2.9 cells/400× stromal field). There was no significant (p = 0.27) decrease in stromal myofibroblasts triggered by topical PRM-151 treatment (11.8 ± 6.6 cells/400× stromal field) compared to the topical vehicle treatment (14.2.8 ± 6.2 cells/400× stromal field). PRM-151 inhibits myofibroblast generation when administered by sub-conjunctival injection, but not when administered topically, after opacity-producing corneal injury. This study provides additional confirmation that bone marrow-derived cells contribute to corneal myofibroblast generation.

Stromal fibroblast-bone marrow-derived cell interactions: implications for myofibroblast development in the cornea.

The purpose of this study was to test the hypothesis that mouse corneal stromal fibroblast and bone marrow-derived cell interactions augment corneal myofibroblast generation and, if so, to study whether such interactions are mediated by paracrine or juxtacrine mechanisms. Mouse bone marrow-derived cells and mouse corneal stromal fibroblasts were obtained from both mice with green fluorescent protein (GFP) expressed in all cells and normal GFP- BL6 control mice. To study the interactions of the different cell types, GFP+ cells of one type were co-cultured with GFP- cells of the other type in Primaria plates (to monitor juxtacrine signaling) or Transwell System plates (to monitor paracrine effects mediated by soluble mediators). Both cell types were cultured at a cell density of 1 × 10(5) cells per ml. The percentage of alpha smooth muscle actin+ myofibroblasts was significantly higher (ANOVA, p<0.001) when bone marrow-derived cells and mouse corneal stromal fibroblasts were co-cultured compared to when bone marrow-derived cells and mouse corneal stromal fibroblasts were cultured alone (control). The in vitro studies using GFP+ corneal fibroblasts or GFP+ bone marrow-derived cells demonstrated conclusively that both cells types could transform into myofibroblasts. However, the percentage of alpha smooth muscle actinassds+ myofibroblasts generated from either cell type precursor was higher when both cells were co-cultured together (juxtacrine) as compared to when bone marrow-derived cells and mouse corneal stromal fibroblasts were co-culture in different compartments of Transwell System (paracrine). Thus, more alpha smooth muscle actin+ GFP+ myofibroblasts were generated from GFP+ corneal stromal fibroblasts when GFP- bone marrow-derived cells were present and more alpha smooth muscle actin+ GFP+ myofibroblasts were generated from GFP+ bone marrow-derived cells when GFP- corneal stromal fibroblasts were present. Polyclonal anti-human latency associated peptide (LAP) (transforming growth factor-β1) neutralizing antibody (a-LAP) and/or transforming growth factor-β type I receptor kinase inhibitor (LY-364947) inhibited the generation of alpha smooth muscle actin+ myofibroblasts from either precursor cell in Transwell System co-culture experiments. These data suggest that TGFβ is a paracrine modulator that regulates the generation of myofibroblasts from either corneal fibroblasts or bone marrow-derived cell precursors.