ECM stiffness modulates the proliferation but not the motility of primary corneal keratocytes in response to PDGF-BB

Iyer, Krithika; Maruri, Daniel P.; Peak, Kara E.; Schmidtke, David W.; Petroll, Walter M; Varner, Victor D.

doi:10.1016/j.exer.2022.109112

Cited by 9 publications

(16 citation statements)

References 61 publications

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“…For stromal cells, ECM stiffness and biochemical cues interact to regulate cell activities ( Figure 1C ). A compliant microenvironment inhibited proliferation of keratocytes cultured in platelet-derived growth factor BB (PDGF-BB) medium ( Iyer et al, 2022 ). However, reduction of effective ECM stiffness significantly inhibited migration of keratocytes cultured in 10% FBS but had no effect when cultured in PDGF-BB ( Kim et al, 2012 ).…”

Section: The Impact Of Mechanical Cues From the Extracellular Matrixmentioning

confidence: 99%

Unraveling the mechanobiology of cornea: From bench side to the clinic

Shu

Tan

Wang

2022

Front. Bioeng. Biotechnol.

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The cornea is a transparent, dome-shaped structure on the front part of the eye that serves as a major optic element and a protector from the external environment. Recent evidence shows aberrant alterations of the corneal mechano-environment in development and progression of various corneal diseases. It is, thus, critical to understand how corneal cells sense and respond to mechanical signals in physiological and pathological conditions. In this review, we summarize the corneal mechano-environment and discuss the impact of these mechanical cues on cellular functions from the bench side (in a laboratory research setting). From a clinical perspective, we comprehensively review the mechanical changes of corneal tissue in several cornea-related diseases, including keratoconus, myopia, and keratectasia, following refractive surgery. The findings from the bench side and clinic underscore the involvement of mechanical cues in corneal disorders, which may open a new avenue for development of novel therapeutic strategies by targeting corneal mechanics.

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Section: The Impact Of Mechanical Cues From the Extracellular Matrixmentioning

confidence: 99%

Unraveling the mechanobiology of cornea: From bench side to the clinic

Shu

Tan

Wang

2022

Front. Bioeng. Biotechnol.

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“…In this context, the storage or elastic modulus appears to be a pivotal feature, which can be modulated to mimic the G ′ of the natural ECM or to impact the cell fate. 32–34 Despite the several advantages of gels for biomedical applications, only a few studies have focused on controlling the stiffness of bioinspired LMWGs via light irradiation. 35,36…”

mentioning

confidence: 99%

“…In this context, the storage or elastic modulus appears to be a pivotal feature, which can be modulated to mimic the G 0 of the natural ECM or to impact the cell fate. [32][33][34] Despite the several advantages of gels for biomedical applications, only a few studies have focused on controlling the stiffness of bioinspired LMWGs via light irradiation. 35,36 In this contribution, we designed a photo-cleavable low molecular weight gelator based on bioinspired and biocompatible architecture glyconucleo-bolaamphiphile (GNBA).…”

mentioning

confidence: 99%

Light-modulation of gel stiffness: a glyconucleoside based bolaamphiphile as a photo-cleavable low molecular weight gelator

Bansode,

Verget,

Barthélémy

2023

Soft Matter

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“…A recent study has shown that corneal keratocytes respond differently in an increased corneal stiffness environment following a freeze injury in vitro. 273 Furthermore, other studies have shown that increased LOX and transglutaminase-2 mRNA activity is proportional to corneal thinning. 203,220,273 Thus, measuring corneal stiffness may allow to study LOX activity in vivo following sulfur mustard injury.…”

Section: Dissertation Discussionmentioning

confidence: 99%

“…273 Furthermore, other studies have shown that increased LOX and transglutaminase-2 mRNA activity is proportional to corneal thinning. 203,220,273 Thus, measuring corneal stiffness may allow to study LOX activity in vivo following sulfur mustard injury. Corneal hysteresis is a new technique for studying corneal stiffness in human patients.…”

Section: Dissertation Discussionmentioning

confidence: 99%

Mechanistic understanding and potential targets for mustard gas keratopathy

Sinha¹

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Sulfur mustard gas (SM), a vesicating and alkylating agent, has been used as a warfare agent since World War I and recently in Syria. SM exposure causes severe corneal injury, pain, and irreversible blindness. The cornea provides twothirds of the eye's light refraction vital for vision. Clinically, SM rapidly penetrates the cornea after exposure and causes a grade-3 or grade-4 clinical pathology called mustard gas keratopathy (MGK). MGK is characterized by ocular inflammation, loss of epithelial barrier, recurrent epithelial erosions, epithelialstromal separation, haze/fibrosis, and neovascularization in the cornea. Despite the high risk of SM for terrorism and mass obliteration, the pathophysiological mechanisms associated with MGK remain elusive. MGK-related corneal haze/fibrosis results from the transdifferentiation of keratocytes into pathological cellular phenotype called myofibroblasts that cause massive overproduction of collagen during wound healing. Keratocytes and collagens residing in the stroma play an essential role in maintaining corneal transparency and are regulated by cytokines and chemokines. The predominant collagens in the stroma are Type I, V, and VII. Additionally, a unique arrangement of collagen fibrils is required for transparency and corneal function. Collagen fibrils are narrow with uniform diameter and arranged with a high degree of lateral ordering. The mechanisms involving myofibroblast formation and collagen derangement after mustard gas exposure are unknown. Presently, a major knowledge gap is how SM exposure affects myofibroblast generation and collagen fibril arrangement in the wounded cornea. My dissertation fills this knowledge gap through successful testing of a novel central hypothesis that mustard gas exposure to the eye results in corneal haze/fibrosis by excessive production of myofibroblasts via TGFβ/SMAD signaling and alters the distinctive organization of collagen fibrils by regulating LOX activity via PI3K/MAPK signaling in the stroma. The formation of haze/fibrosis post SM exposure was tested using an in vivo rabbit model, in which live New Zealand White rabbits received SM vapor exposure localized to the eye. The goals of my PhD thesis were to characterize time-dependent changes in the stroma in live animals in situ employing eye examinations and state-of-the-art multimodal clinical ophthalmic imaging techniques in live rabbits with Stereo, Slit-lamp, HRT-RCM3, and Spectralis microscopy system. Fantes grading, fluorescein staining, Schirmer's tests, pachymetry, and applanation tonometry were conducted to measure levels of corneal haze, ocular surface healing, tears, corneal thickness, and intraocular pressure, respectively. H and E and PSR staining were used for histopathological cellular changes in the cornea. In vivo confocal and OCT imaging revealed significant changes in structural and morphological appearance of the cornea in vivo in SM-exposed rabbit corneas in a time-dependent manner compared to naive cornea (p [less than] 0.001 or p [less than] 0.0001). Also, SM-exposed eyes showed loss of corneal transparency characterized by increased stromal thickness (p [less than] 0.0001), debris, and light-scattering myofibroblasts or activated keratocytes, representing haze formation in the cornea. Naive eyes did not show any structural, cellular, and functional abnormalities. SM exposure showed increased central corneal thickness, ocular edema and tearing compared to the eyes of the naive (p [less than] 0.001 or p [less than] 0.0001). This study concluded that structural and cellular changes in primary corneal layers are early pathological events contributing to MGK in vivo. Next, I tested if TGFβ/SMAD is a major molecular mechanism for myofibroblast formation following SM exposure. Rabbit corneas exposed to SM had a time-dependent increase in [alpha]SMA, a maker for myofibroblast formation. Additionally, using an established in vitro model of primary human corneal fibroblasts (hCSF) treated with nitrogen mustard (NM), an analogous agent to SM, or NM+SMAD inhibitors, I verified my hypothesis that SMAD is a predominant pathway for myofibroblast formation in MGK. Primary hCSF treated with NM showed a significant time-dependent increase (p [less than] 0.05, p [less than] 0.01) in [alpha]SMA expression which was significantly decreased by SMAD inhibitors (p [less than] 0.05, p [less than] 0.01). This data strongly supported my notion that SMAD is a major pathway for myofibroblast formation in corneal stroma after mustard gas exposure. The final section of my thesis tested another novel postulate that lysyl oxidase (LOX) is a possible mechanism to cause changes in the collagen lamella. LOX activity regulates extracellular cross-linking; however, literature/knowledge on LOX function in the cornea is very limited. Thus, I tested if p38 and PI3K are major regulatory pathways. p38 and PI3K have shown to regulate steady state LOX mRNA in cardiac fibroblasts. An in vitro study using hCSF cells, NM, p38 inhibitor, and PI3K inhibitor concluded that (a) p38 follow the pattern similar to SMAD signaling, and (b) PI3K was directly related to LOX production. Subsequent research showed that p38 has a function correlative to SMAD function in the corneal epithelium. This finding was the first to identify the role of PI3K in the mustard gas exposed corneal stroma. In conclusion, my dissertation identified time-dependent in situ structural and cellular aberrations in the rabbit cornea in vivo after mustard gas exposure and remarkably improved mechanistic knowledge that will be instrumental in developing novel strategies and molecular targets for newer therapeutics to prevent and treat corneal mustard gas keratopathy. Additionally, it might help understanding corneal pathologies caused by other alkylating/vesicating agents.

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ECM stiffness modulates the proliferation but not the motility of primary corneal keratocytes in response to PDGF-BB

Cited by 9 publications

References 61 publications

Unraveling the mechanobiology of cornea: From bench side to the clinic

Unraveling the mechanobiology of cornea: From bench side to the clinic

Light-modulation of gel stiffness: a glyconucleoside based bolaamphiphile as a photo-cleavable low molecular weight gelator

Mechanistic understanding and potential targets for mustard gas keratopathy

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