Name: Video: Collection of Subendothelial Matrix from Retinal Microvascular Endothelial Cell (REC) Cultures for Atomic Force Microscope (AFM) Stiffness Measurement
Uploaded: 2024-07-12T14:30:00
Description: 32 Views. Collection of Subendothelial Matrix from Retinal Microvascular Endothelial Cell (REC) Cultures for Atomic Force Microscope (AFM) Stiffness Measurement

collection of subendothelial matrix from retinal microvascular endothelial cell (rec) cultures for atomic force microscope (afm) stiffness measurement

Measurement of Stiffness in Mouse Retinal Capillaries and Subendothelial Matrix

Retinal capillaries are essential for maintaining retinal homeostasis and visual function. Indeed, their degeneration in early diabetes is strongly implicated in the development of vision-threatening complications of diabetic retinopathy (DR), a microvascular condition that affects nearly 40% of all individuals with diabetes1. Vascular inflammation contributes significantly to retinal capillary degeneration in DR. Past studies have demonstrated an important role for aberrant molecular and biochemical cues in diabetes-induced retinal vascular inflammation2,3. However, recent work has introduced a new paradigm for DR pathogenesis that identifies retinal capillary stiffening as a crucial yet previously unrecognized determinant of retinal vascular inflammation and degeneration4,5,6.
Specifically, the diabetes-induced increase in retinal capillary stiffness is caused by the upregulation of collagen crosslinking enzyme lysyl oxidase (LOX) in retinal endothelial cells (ECs), which stiffens the subendothelial matrix (basement membrane)4,5,6. Matrix stiffening, in turn, stiffens the overlying retinal ECs (due to mechanical reciprocity), thus leading to the overall increase in retinal capillary stiffness4. Crucially, this diabetes-induced retinal capillary stiffening alone can promote retinal EC activation and inflammation-mediated EC death. This mechanical regulation of retinal EC defects can be attributed to altered endothelial mechanotransduction, the process by which mechanical cues are converted into biochemical signals to produce a biological response7,8,9. Importantly, altered EC&#160;mechanical cues and subendothelial matrix structure have also been implicated in choroidal vascular degeneration associated with early age-related macular degeneration (AMD)10,11,12, which attests to the broader implications of vascular mechanobiology in degenerative retinal diseases.
Notably, retinal capillary stiffening occurs early on in diabetes, which coincides with the onset of retinal inflammation. Thus, the increase in retinal capillary stiffness may serve as both a therapeutic target and an early diagnostic marker for DR. To this end, it is important to obtain reliable and direct stiffness measurements of retinal capillaries and subendothelial matrix. This can be achieved by using an atomic force microscope (AFM), which offers a unique, sensitive, accurate, and reliable technique to directly measure the stiffness of cells, extracellular matrix, and tissues13. An AFM applies minute (nanoNewton-level) indentation force on the sample whose stiffness determines the extent to which the indenting AFM cantilever bends- the stiffer the sample, the more the cantilever bends, and vice versa. We have used AFM extensively to measure the stiffness of cultured endothelial cells, subendothelial matrices, and isolated mouse retinal capillaries4,5,6,11,12. These AFM stiffness measurements have helped identify endothelial mechanobiology as a key determinant of DR and AMD pathogenesis. To help broaden the scope of mechanobiology in vision research, here we provide a step-by-step guide on the use of AFM for stiffness measurements of isolated mouse retinal capillaries and subendothelial matrix.

Author Spotlight: Investigating Vascular Stiffness and Inflammation in Early Diabetic Retinopathy

Isolation of Mouse Retinal Capillaries and Subendothelial Matrix for Stiffness Measurement Using Atomic Force Microscopy

Collection of Subendothelial Matrix from Retinal Microvascular Endothelial Cell (REC) Cultures for Atomic Force Microscope (AFM) Stiffness Measurement

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Research

JoVE Journal

Biology

32 Views. Collection of Subendothelial Matrix from Retinal Microvascular Endothelial Cell (REC) Cultures for Atomic Force Microscope (AFM) Stiffness Measurement

Video: Collection of Subendothelial Matrix from Retinal Microvascular Endothelial Cell (REC) Cultures for Atomic Force Microscope (AFM) Stiffness Measurement

Retinal capillary degeneration is a clinical hallmark of the early stages of diabetic retinopathy (DR). Our recent studies have revealed that diabetes-induced retinal capillary stiffening plays a crucial and previously unrecognized causal role in inflammation-mediated degeneration of retinal capillaries. The increase in retinal capillary stiffness results from the overexpression of lysyl oxidase, an enzyme that crosslinks and stiffens the subendothelial matrix. Since tackling DR at the early stage is expected to prevent or slow down DR progression and associated vision loss, subendothelial matrix, and capillary stiffness represent relevant and novel therapeutic targets for early DR management. Further, direct measurement of retinal capillary stiffness can serve as a crucial preclinical validation step for the development of new imaging techniques for non-invasive assessment of retinal capillary stiffness in animal and human subjects. With this view in mind, we here provide a detailed protocol for the isolation and stiffness measurement of mouse retinal capillaries and subendothelial matrix using atomic force microscopy.

We recently identified retinal capillary stiffening as a new paradigm for retinal dysfunction associated with diabetes. This protocol elaborates the steps for isolation of mouse retinal capillaries and the subendothelial matrix from retinal endothelial cultures, followed by a description of the stiffness measurement technique using atomic force microscopy.