Collagen XIV Is an Intrinsic Regulator of Corneal Stromal Structure and Function

Abstract

Collagen XIV is poorly characterized in the body, and the current knowledge of its function in the cornea is limited. The aim of the current study was to elucidate the role(s) of collagen XIV in regulating corneal stromal structure and function. Analysis of collagen XIV expression, temporal and spatial, was performed at different postnatal days (Ps) in wild-type C57BL/6 mouse corneal stromas and after injury. Conventional collagen XIV null mice were used to inquire the roles that collagen XIV plays in fibrillogenesis, fibril packing, and tissue mechanics. Fibril assembly and packing as well as stromal organization were evaluated using transmission electron microscopy and second harmonic generation microscopy. Atomic force microscopy was used to assess stromal stiffness. Col14a1 mRNA expression was present at P4 to P10 and decreased at P30. No immunoreactivity was noted at P150. Abnormal collagen fibril assembly with a shift toward larger-diameter fibrils and increased interfibrillar spacing in the absence of collagen XIV was found. Second harmonic generation microscopy showed impaired fibrillogenesis in the collagen XIV null stroma. Mechanical testing suggested that collagen XIV confers stiffness to stromal tissue. Expression of collagen XIV is up-regulated following injury. This study indicates that collagen XIV plays a regulatory role in corneal development and in the function of the adult cornea. The expression of collagen XIV is recapitulated during wound healing.

Figure 1

Col14a1 expression in the cornea decreases with maturation, and collagen XIV expression weakens in early adulthood. A:Col14a1 mRNA expression (pink) in the mouse cornea decreases after postnatal day (P) 4, shown by normalized Col14a1 mRNA expression in real-time PCR analysis. B: Protein expression of collagen XIV was decreased at P30 and mostly absent at P150. Left panel: The representative WES images of collagen XIV expression and total protein. Right panel: The quantitation of the collagen XIV normalized with total protein. The error bars represent the SD from two independent experiments. Three to five corneas of different mice at each age were used. Experiments were repeated three times. ∗P < 0.05, ∗∗∗P < 0.001.

Figure 2

Collagen XIV localizes throughout the entire corneal stroma (St). Collagen XIV (red) localizes throughout the entire corneal stroma during development from postnatal day (P) 4 and P30. Immunoreactivity decreases at P30 and mostly localizes to the anterior stroma at P90 and P150. A P4 Col14a1^−/− cornea is used as the negative control. n = 3. Scale bars = 50 μm. En, endothelium; Ep, epithelium.

Figure 3

Composite images show collagen XIV expression persistence in the scleral stroma of adult eyes. Collagen XIV expression in wild-type C57BL/6 eye is persistent in the adult sclera (S), whereas corneal expression is rapidly decreasing. n = 3. Scale bars = 450 μm. K, cornea; L, limbal region; P, postnatal day.

Figure 4

Second harmonic imaging of intact eye demonstrates impaired stromal compaction and suggests impaired fibrillogenesis in the absence of collagen XIV. Forward-scatter signal in wild-type C57BL/6 (WT) corneas at different ages (leftcolumn) compared with Col14a1^−/− corneas (rightcolumn) shows stronger intensity in the WT corneas. n = 4. Scale bars = 25 μm. En, endothelium; Ep, epithelium; P, postnatal day; St, stroma.

Figure 5

Collagen XIV is a regulator of fibril assembly during stromal maturation. A and B: Collagen fibril diameters are slightly larger in the absence of collagen XIV (B) than in the wild-type C57BL/6 (WT) corneal stroma (A). C: The mean fibril diameter in the C57BL/6 cornea is 30.7 ± 3.8 nm, and fibrils have a mean diameter of 32 ± 3.5 nm in the absence of collagen XIV. D: The fibril density decreases significantly in Col14a1^−/− stromas. n = 3 (both groups). ∗∗∗P < 0.001. Scale bars = 200 nm (A and B).

Figure 6

Collagen XIV influences corneal stiffness by increasing tissue resistance to compression. A: Wild-type C57BL/6 (WT) stromas were more resistant to compression than the Col14a1^−/− stromas, with a significant decrease in Young's modulus and corneal stiffness without collagen XIV. B: The distribution of Young's modulus in Col14a1^–/– corneas was narrower than that in WT corneas. Sixteen corneas were used in this experiment, eight WT and eight Col14a1^−/−. ∗∗P < 0.01.

Figure 7

Collagen XIV expression is recapitulated following stromal injury. A and B: Images demonstrating loss of collagen XIV expression at 1 week, shown by arrow (A), followed by re-expression of collagen XIV, shown by asterisks, at 3 weeks (B). C: Real-time PCR shows up-regulation of Col14a1 mRNA expression during wound healing. D: Heat map demonstrates the effects of collagen XIV in influencing matrix composition by fibroblasts expanded in vitro; protein from 40 corneas was used in this experiment, and 20 wild-type C57BL/6 (WT) and 20 Col14a1^−/− were used for in vitro expansion. n = 6 (A and B); n = 3 (C). ∗P < 0.05. En, endothelium; Ep, epithelium; St, stroma.