Effective Gene Delivery to Valvular Interstitial Cells Using Adeno-Associated Virus Serotypes 2 and 3.

Currently, curative therapies for heart valve diseases do not exist, thus motivating the need for new therapeutics, regenerative and tissue-engineered valves, and further basic research into pathological mechanisms. For studying valve diseases and developing valve therapies, effective methods to manipulate gene expression in primary valvular interstitial cells (VICs), which promote calcification in disease, would be valuable. Unfortunately, there is little information reported about effective gene delivery methods for VICs. Adeno-associated virus (AAV) is a clinically proven gene delivery vector capable of transducing many cell types and tissues, but has not yet been reported to infect valvular cells. In this study, AAV serotypes 1-9 were tested for their ability to deliver a green fluorescent protein (GFP) reporter into VICs in vitro. Flow cytometry results indicate AAV2 and AAV3 are capable of transducing VICs more efficiently than other serotypes. Furthermore, transduction efficiencies can be optimized by increasing the multiplicity of infection (MOI) and using self-complementary, double-stranded genomes, yielding up to 98% successfully transduced cells. Transduction of VICs by AAV2 or AAV3 in the presence of competing soluble heparin significantly reduces delivery efficiencies, suggesting heparan sulfate proteoglycans act as the primary VIC receptors of these two serotypes. Overall, this study establishes AAV2 and AAV3 as efficient gene delivery vehicles for primary VICs. Such effective delivery vectors for valve cells may be broadly useful for numerous applications, including the study of valvular cell biology, development of valve disease therapies, and regulation of genes for tissue engineering heart valves.

Whole globe inflation testing of exogenously crosslinked sclera using genipin and methylglyoxal.

Exogenous collagen cross-linking has been investigated as method of reinforcing scleral biomechanics, with the goal of counteracting scleral weakening that occurs at the onset of myopia. This study uses whole globe inflation testing to investigate the biomechanical effect of treating posterior sclera with the collagen cross-linking agents methylglyoxal and genipin. Pairs of porcine eyes were treated in four ways. Three groups involved 1% methylglyoxal: two-hour (Group I) or thirty-minute (Group II) incubation of the whole globe, and thirty-minute incubation of only the posterior sclera of the intact eye (Group III). Group IV consisted of a thirty-minute incubation of the posterior sclera in 1% genipin. Following treatment, each eye was subjected to inflation testing under physiological pressure levels (0-150 mmHg); four strain markers on the posterior pole were tracked, providing displacement measurements in two directions. Results were used to derive load versus deformation behavior and to calculate stiffness at 0.25% strain (toe stiffness) and at peak strain (peak stiffness). Toe stiffness of Group I was 4.8 and 1.3 times greater than controls (sagittal and transverse directions, respectively: 5.23 ± 0.39 vs. 0.90 ± 0.08 mHg, P < 0.001; and 3.41 ± 0.19 vs. 1.51 ± 0.22 mHg, P < 0.01; values in mean ± SE). Group II was 7.4 and 4.3 times stiffer than controls (sagittal and transverse directions, respectively: 5.26 ± 0.49 vs. 0.63 ± 0.10 mHg, P < 0.02; and 3.44 ± 0.44 vs. 0.65 ± 0.07 mHg, P < 0.003). Group III was 3.6 and 3.4 times stiffer than controls (sagittal and transverse directions, respectively: 5.21 ± 0.39 vs. 1.13 ± 0.31 mHg, P < 0.01; and 4.94 ± 1.48 vs. 1.13 ± 0.25, P < 0.01), while Group IV was 8.2 and 2.8 times stiffer than controls (sagittal and transverse: 12.36 ± 1.96 vs. 1.35 ± 0.14 mHg, P < 0.01; and 12.45 ± 1.34 vs. 3.27 ± 0.50 mHg, P < 0.05). In all groups, there was no significant difference in peak stiffness after scleral cross-linking (SXL). At low strain, the posterior sclera was stiffer in both measured directions following methylglyoxal and genipin treatments, however at peak strain the treated sclera was not stiffer. Additionally, the saturation level of scleral stiffening by methylglyoxal can be reached within thirty minutes of treatment.

Cross-linking with ultraviolet-a and riboflavin reduces corneal permeability.

PURPOSE: To investigate the effect of cross-linking treatment on corneal permeability in a live animal model. METHODS: Rabbit eyes were selected at random to be left unoperated or to undergo epithelial debridement with or without treatment consisting of cross-linking (CXL) with riboflavin and ultraviolet-A. Nine eyes received a total dose of 3.6 J/cm² and after epithelial healing the corneas were placed in a two-chamber system for quantification of the diffusion of fluorescein compared with controls. Thirty eyes received a total dose of 5.4 J/cm² and, after epithelial healing, in vivo corneal permeability was quantified as the pupillary response over a 30-minute period to a dose of topical pilocarpine compared with controls. RESULTS: In the ex vivo assay, the mean permeability coefficient in the CXL group (2.42 × 10⁻7) was reduced when compared with the unoperated controls (3.73 × 10⁻7; P = 0.007) and to the eyes that received epithelial debridement alone (3.74 × 10⁻7; P = 0.01). In the in vivo permeability assay, the change in pupillary diameter at 30 minutes after pilocarpine administration was smaller in the CXL group (-1.9 mm), compared with the epithelial debridement group (-2.6 mm; P < 0.001) and with the unoperated controls (-2.7 mm; P = 0.003). CONCLUSIONS: Corneal cross-linking with ultraviolet-A and riboflavin results in a statistically significant reduction in corneal permeability. These findings suggest that dosing of topical medications may need to be increased in eyes with a history of CXL to achieve expected therapeutic effects, and they may have implications for the long-term health of the cornea.