Acute hypoxia influences collagen and matrix metalloproteinase expression by human keratoconus cells <i>in vitro</i>

<div><p>Keratoconus (KC) is a progressive corneal ectasia linked to thinning of the central cornea. Hard contact lenses, rigid gas permeable lenses, and scleral lenses are the primary treatment modalities for early to mid- stages of KC to correct refractive error and astigmatism that develops as a result of an irregular corneal structure. These treatments are associated with significant drawbacks, including reduced availability of the tear film and oxygen to the corneal epithelium and stroma. However, it remains unknown whether hypoxia affects corneal integrity in the KC pathobiology. A number of studies have associated elevated oxidative stress with KC both <i>in vitro</i> and <i>ex vivo</i>. We hypothesized that KC-derived corneal fibroblasts are more susceptible to hypoxia-induced oxidative stress compared to healthy controls leading to exacerbation of corneal thinning in KC. This study investigated the effects of hypoxia on ECM secretion, assembly, and matrix metalloproteinase (MMP) expression in human corneal fibroblasts from healthy controls (HCFs) and KC patients (HKCs) <i>in vitro</i>. HCFs and HKCs were cultured in 3D constructs for 3 weeks and maintained or transferred to normoxic (21% O₂) or hypoxic (2% O₂) conditions, respectively, for 1 additional week. At the 4 week time-point, constructs were isolated and probed for Collagen I, III, and V, keratocan and MMP-1, -2, -3, -9, and -13, as well as hypoxia markers, hypoxia inducible factor-1α and lactoferrin. Conditioned media was also collected and probed for Collagen I, III, and V by Western blot. Thickness of the ECM assembled by HCFs and HKCs was measured using immunofluorescence microscopy. Results showed that hypoxia significantly reduced Collagen I secretion in HKCs, as well as upregulated the expression of MMP-1 and -2 with no significant effects on MMP-3, -9, or -13. ECM thickness was reduced in both cell types following 1 week in a low oxygen environment. Our study shows that hypoxia influences collagen and MMP expression by HKCs, which may have consequential effects on ECM structure in the context of KC.</p></div

AZGPI quantified mRNA expression for A) HCF and B) HKC cell types.

<p>Four conditions were tested: Control, TGF-β1, TGF-β2, and TGF-β3. In HCFs all three TGFβ isoforms resulted in significant down regulation of AZGPI expression. TGFβ1 showed the lowest of the three when compared to the controls. In HKCs, no significance changes in AZGPI expression was shown for TGFβ1 and TGFβ2 isoforms. TGFβ3 stimulation significantly down regulated AZGPI protein expression. All error bars represent standard error (SEM) of n = 3.</p

Pentacam data collected from participating subjects.

<p>Summary of the mean ages, corneal thickness (Ct min) and maximum keratometric (Kmax) values for: Controls and Keratoconus. Mean age for Control was 33 and for KC the average age was 30. No statistically significant differences were found. The Ct min for Controls was 501.2 and KC was recorded as 466. There was a statistically difference between the two groups (p<0.05). Kmax average value for the controls was 46.9 and the KC average value was 53.9.</p><p>Pentacam data collected from participating subjects.</p

Selected proteins regulated in tears. * indicates statistical significance.

<p>Summary of the most common proteins regulated in KC tear samples. Lipophilin-A, Immunoglobulin J Chain, Cystatin-S, and Lactotransferrin were all significantly down regulated (p<0.0001) in KC tear samples. No significant changes in the levels of Secreted frizzled-related protein 1 and AZGPI (p<0.076 and p = 0.18 respectively) was found. PIP was significantly down regulated (p<0.0001) in KCs.</p><p>Selected proteins regulated in tears. * indicates statistical significance.</p

PIP quantified mRNA expression for A) HCF and B) HKC cell types.

<p>Four conditions were tested: Control, TGF-β1, TGF-β2, and TGF-β3. All three TGF-β isoforms resulted in significant down regulation of PIP for both cell types. All error bars represent standard error (SEM) of n = 3.</p

PIP quantified protein expression for A) HCF and B) HKC cell types.

<p>Four conditions were tested: Control, TGF-β1, TGF-β2, and TGF-β3. HCFs expression was increased upon TGFβ treatments however none of them reached significance. HKCs showed significant down regulation upon all TGFβ treatments. TGFβ3 showed the lowest PIP protein levels compared to all other conditions. HKCs PIP protein levels were consistently lower under all conditions when compared to HCFs. <u>Note</u>: HCF’s PIP expression was seen at 17 kDa protein whereas in HKC’s at 37 kDa. All error bars represent standard error (SEM) of n = 3.</p