Synthesis and Characterization of a Novel Peptide Targeting Human Tenon Fibroblast Cells To Modulate Fibrosis: An Integrated Empirical Approach

Fibrosis is the primary factor influencing the prognosis of glaucoma post-trabeculectomy surgery, an eye condition characterized by increased intraocular pressure (IOP). Despite advancements in surgical procedures and aftercare, it continues to be a serious impediment. During the clinical intervention of scarring, fibrosis is managed by using topical application of combined antifibrotic drugs (mitomycin C). But still, scarring remains a key problem due to minimal drug penetration and nonbioavailability. In this study, we synthesized a cell-specific peptide for modulating scarring in human tenon fibroblasts undergoing epithelial–mesenchymal transition (EMT). The peptide was also conjugated with mitomycin C in order to investigate the effect of the drug conjugation on human tenon fibroblasts from the nanofiber composite system and to evaluate the fibrosis process. Peptide VRF2019 was identified using a subtractive proteomics approach, including solubility, cell penetration, and amphipathic properties. The peptide structure was determined using circular dichroism spectroscopy. The peptide and drug was conjugated using N-ethyl-N′-(3-(dimethylamino)propyl) carbodiimide/N-hydroxysuccinimide (EDC-NHS) chemistry, and the conjugation efficiency was evaluated using high-pressure liquid chromatography. The conjugated product and polycaprolactone (PCL) were electrospun to form a composite nanofiber, which was tested for cytotoxicity and drug release on human tenon fibroblast cells. The modeled VRF2019 peptide structure formed an α-helical structure with all residues spanning the allowed regions of the Ramachandran plot. Subsequent molecular dynamics simulations also demonstrated its membrane penetration potential. The peptide uptake was also studied in human tenon fibroblast cells. High-pressure liquid chromatography (HPLC) and mass spectrometry measurements confirmed peptide–drug conjugation and stability. Furthermore, scanning electron microscopy (SEM) investigation revealed the structure and size of the PCL composite nanofiber. We infer from early research that the PCL composite nanofiber matrix can greatly increase drug delivery and bioavailability

Genetic Association of SNPs near <i>ATOH7</i>, <i>CARD10</i>, <i>CDKN2B</i>, <i>CDC7</i> and <i>SIX1/SIX6</i> with the Endophenotypes of Primary Open Angle Glaucoma in Indian Population

<div><p>Primary open angle glaucoma (POAG) belonging to a group of optic neuropathies, result from interaction between genetic and environmental factors. Study of associations with quantitative traits (QTs) is one of the successful strategies to understand the complex genetics of POAG. The current study attempts to explore the association of variations near/in genes like <i>ATOH7</i>, <i>SIX1/SIX6</i> complex, <i>CDKN2B</i>, <i>CARD10</i>, and <i>CDC7</i> with POAG and its QTs including vertical cup to disc ratio (VCDR), central corneal thickness (CCT), intra ocular pressure (IOP), and axial length (AL). Case-control study design was carried out in a sample size of 97 POAG cases and 371 controls from South India. Model-based (additive, recessive, dominant) association of the genotypes and their interaction was carried out between cases and controls using chi-square, linear and logistic regression methods. Nominal significance (<i>P</i><0.05) was observed for QTs like i) VCDR with SNPs rs1900004 (<i>ATOH7</i>); rs1192415 (<i>CDC7)</i>; rs10483727 (<i>SIX1/SIX6)</i>, rs9607469 (<i>CARD10);</i> ii) CCT with rs1192415; iii) IOP with rs1900004 and iv) AL with rs1900004 and rs1063192 (<i>CDKN2B)</i>. We were able to replicate previously known interactions between <i>ATOH7-SIX6</i> and <i>SIX6-CDKN2B</i> along with few novel interactions between <i>ATOH7</i>—<i>CDC7</i> and <i>SIX6</i> with genes including <i>CARD10</i> and <i>CDC7</i>. In summary, our results suggest that a probable interaction among the candidate genes for QTs, play a major role in determining the individual’s susceptibility to POAG.</p></div

Multiple linear regression analysis for the SNPs with AL, CCT, VCDR and IOP as dependant variable in the whole cohort.

<p>^Ω = <i>P(</i>β± SE)</p><p>β = standard regression coefficient; SE = standard error; AL-axial length; CCT- Central corneal thickness, VCDR-vertical cup to disc ratio, IOP- intra ocular pressure.</p><p>*Obtained from linear regression, adjusted for age and gender</p><p>Bonferroni adjusted significant level< = 0.008 (0.05/6). Variation in no of samples depends on the availability of data.</p><p>Multiple linear regression analysis for the SNPs with AL, CCT, VCDR and IOP as dependant variable in the whole cohort.</p

Analysis of minor allele frequencies and χ^<b>2</b> of the SNPs between POAG and controls.

<p>M- Major allele, m-minor allele</p><p>* <i>P</i> values are derived from χ² tests; significance: P<0.05</p><p>Analysis of minor allele frequencies and χ^<b>2</b> of the SNPs between POAG and controls.</p

Association of the SNPs with CCT, VCDR, IOP, AL in controls and POAG (HTG/NTG/combined).

<p>β = standard regression coefficient</p><p>SE = standard error</p><p>£- dominant model</p><p>∞- recessive model</p><p>¶-additive model</p><p>*Obtained from linear regression</p><p>Bonferroni adjusted significant level< = 0.008 (0.05/6)</p><p>Association of the SNPs with CCT, VCDR, IOP, AL in controls and POAG (HTG/NTG/combined).</p

Demographic and clinical features of the study subjects.

<p>*the age at recruitment for controls or age of disease onset for POAG patients are shown</p><p>SD-standard deviation, IOP- intraocular pressure, VCDR-vertical cup to disc ratio, CCT- central corneal thickness, AL- axial length, ACD- anterior chamber depth.</p><p>Demographic and clinical features of the study subjects.</p

Association analysis between <i>ABCC5</i> rs1401999 and primary angle closure glaucoma in all chip-typed sample collections (top panel), de-novo genotyped sample collections (middle panel), and PACG cases and clinically certified controls with open angles (bottom panel).

<p>MAF case: Minor allele frequency in PACG cases.</p><p>MAF control: Minor allele frequency in controls.</p><p>OR: Odds ratio.</p><p><i>P</i>: <i>P</i>-value for association with PACG.</p><p>I²: I-squared index for between-collection heterogeneity.</p><p>* Results here are presented based on raw minor allele frequency counts without further adjustment.</p>†<p>PACG patients were recruited from the Beijing Tongren Hospital and controls were recruited from the Handan Eye Study (HES), a population-based study of eye disease in rural Chinese aged 30 years and over.</p

Association analysis between <i>ABCC5</i> rs1401999 and susceptibility to primary angle closure glaucoma (PACG).

<p>The PACG sample collections have been described elsewhere <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004089#pgen.1004089-Vithana1" target="_blank">[6]</a>. The vertical line represents a per-allele odds ratio of 1.00. The oblongs represent point estimates (referring to the per-allele odds ratio), with the height of the oblongs inversely proportional to the standard error of the point estimates. Horizontal lines indicate the 95% confidence interval for each point estimate. Meta-analyses of samples are reflected by blue diamonds. The width of the diamonds indicates their 95% confidence intervals. All point estimates in Stage 1 have been adjusted for the top axes of genetic stratification using logistic regression.</p

Quantitative trait analysis between <i>ABCC5</i> rs1401999 and anterior chamber depth in SIMES, SINDI, and BES.

<p>SIMES: Singapore Malay Eye Study (typed with Illumina 610K GWAS chip).</p><p>SINDI: Singapore Indian Eye Study (typed with Illumina 610K GWAS chip).</p><p>BES1: Beijing Eye Study typed with Illumina 610K GWAS chip.</p><p>BES2: Beijing Eye Study typed with direct sequencing.</p><p>β: Per-allele effect size of <i>ABCC5</i> rs1401999 on anterior chamber depth.</p><p>SE: Standard error for β.</p><p><i>P</i>gc: Genomic control corrected <i>P</i>-value.</p><p>MAF: Minor allele frequency.</p><p>*: I²-index for heterogeneity = 0%.</p