Alpha-Smooth Muscle Actin Expression and Parafoveal Blood Flow Pathways Are Altered in Preclinical Diabetic Retinopathy

Abstract

Purpose: To investigate differences in alpha smooth muscle actin (αSMA) expression and parafoveal blood flow pathways in diabetic retinopathy (DR).

Methods: Human donor eyes from healthy subjects (n = 8), patients with diabetes but no DR (DR-; n = 7), and patients with clinical DR (DR+; n = 13) were perfusion labeled with antibodies targeting αSMA, lectin, collagen IV, and filamentous actin. High-resolution confocal scanning laser microscopy was used to quantify αSMA staining and capillary density in the parafoveal circulation. Quantitative analyses of connections between retinal arteries and veins within the superficial vascular plexus (SVP), intermediate capillary plexus (ICP) and deep capillary plexus (DCP) were performed.

Results: Mean age between the groups was not different (P = 0.979). αSMA staining was seen in the SVP and ICP of all groups. The DCP was predominantly devoid of αSMA staining in control eyes but increased in a disease stage-specific manner in the DR- and DR+ groups. The increase in αSMA staining was localized to pericytes and endothelia of terminal arterioles and adjacent capillary segments. Capillary density was less in the DCP in the DR+ group (P < 0.001). ICP of the DR- and DR+ groups received more direct arteriole supplies than the control group (P < 0.001). Venous outflow pathways were not altered (all P > 0.284).

Conclusions: Alterations in αSMA and vascular inflow pathways in preclinical DR suggest that perfusion abnormalities precede structural vascular changes such as capillary loss. Preclinical DR may be characterized by a "steal" phenomenon where blood flow is preferentially diverted from the SVP to the ICP and DCP.

Figure 1.

Comparison of αSMA staining distribution between an artery (a) and a vein (v) across the control group (A), DR– group (B), and DR+ group (C). Strong αSMA staining of the artery, arterioles, and capillaries adjacent to the artery was observed for all three groups. In the control group, αSMA staining was largely devoid from venules but demonstrated strong staining at venular junction sites (green arrows). In the DR– group, additional αSMA staining was observed at venular junctions (green arrows) and in some short capillary segments (orange arrows). In the DR+ group, significantly more αSMA staining was found on the venous aspect of the vasculature. Some capillaries had more αSMA staining across an entire segment (orange arrows). Significantly more αSMA staining was seen along the distal portion of the vein and all of its tributaries (green arrow). A microaneurysm was seen adjacent to an area of capillary non-perfusion (white asterisk). αSMA, F-actin, and nucleus were false-colored yellow, red, and blue, respectively. All images are to the same scale. Scale bars: 150 µm.

Figure 2.

Comparisons of the superficial vascular plexus among the control group (A, B), DR– group (C, D), and DR+ group (E, F). All images were acquired from within the macula region, superior to the fovea. The left panel contains images with collagen IV staining, and the right panel contains the same images with αSMA staining. Consistent staining patterns were observed for collagen IV across the three groups, where all orders of vessels demonstrated similar staining intensities. The a4 and v4 vessels are indicated on the collagen IV panel. For αSMA staining, retinal veins from both the DR– and DR+ groups demonstrated stronger staining compared to the control group. Arteries are indicated by red arrows and veins by green arrows in both panels. All images are to the same scale. Scale bar: 300 µm.

Figure 3.

Comparisons of the intermediate capillary plexus among the control group (A, B), DR– group (C, D), and DR+ group (E, F). The left panel contains images with collagen IV staining, and the right panel contains the same images with αSMA staining. Collagen IV staining patterns were similar between the control and DR– groups. In the DR+ group, a microaneurysm can be seen (red arrow). There was more αSMA staining in the DR– and DR+ groups compared to the control group. Many capillaries of the DR+ group appeared hyperfluorescent (green arrows). All images are to the same scale. Scale bar: 300 µm.

Figure 4.

Comparisons of the deep capillary plexus among the control group (A, B), DR– group (C, D), and DR+ group (E, F). The left panel contains images with collagen IV staining, and the right panel contains the same images with αSMA staining. Collagen IV staining patterns were similar between the control and DR– groups. The DR+ group demonstrated several capillary non-perfusion sites (yellow asterisks) and microaneurysms (red arrows). Levels of αSMA staining across the three groups differed, as minimal αSMA staining was seen in the control group (B), but more αSMA staining was found in the DR– group. There was evidence of focal hyperfluorescence of capillary segments (D; green arrows). The DR+ group showed significantly more αSMA staining, with many capillary segments showing hyperfluorescence (F; green arrows). The microaneurysm seen in the DR+ panel is highlighted in an inset. All images are to the same scale. Scale bar: 300 µm.

Figure 5.

Illustration of αSMA staining patterns of the DCP in the control group using three-dimensional (3D) high-resolution microscopy. (A) The 3D stack shows the connections between the ICP and the DCP via pre-capillary arterioles designated as a1. (B) The projected view of all slices illustrates the sites of αSMA termination as a1 enters the DCP; pericytes that expressed αSMA can be seen along the a1 (green arrow). (C) The spatial relationship between an endothelial cell and the pericyte along a1. (D) αSMA expression by the pericyte. (E, F) Endothelial cells of the more distal segment of DCP capillary did not express αSMA (magenta arrows). The pericyte also did not express αSMA (green arrows). αSMA, F-actin, and nucleus were false-colored yellow, red, and blue, respectively. Scale bars: 30 µm.

Figure 6.

αSMA staining patterns of the DCP in the DR– group. (A, B) αSMA staining can be seen within a1 and the proximal segment of capillary (c) downstream of a1. (C) The transition sites of αSMA expression/non-expression within DCP capillaries are indicated by blue arrows; panel C is an inset of A and B. (D) A capillary segment of interest is marked by a green asterisk. A pericyte that expressed αSMA is indicated by a green arrow. (E) Magnified view of this pericyte, as well as an αSMA-expressing endothelial cell (magenta arrow indicates an elongated cell nucleus). Beyond this endothelial cell, the capillary no longer expressed αSMA. αSMA, F-actin, and nucleus were false-colored yellow, red, and blue, respectively. Scale bars: 100 µm.

Figure 7.

αSMA staining patterns of the DCP in the DR+ group. (A) The points of inflow (a1) and outflow (v3) are marked on a DCP section labeled with F-actin. (B) There was significantly increased αSMA staining in the DCP. (C) Sites of increased αSMA staining included capillary segments adjacent to a1 (magenta arrows) and v3 (green arrows). αSMA, F-actin, and nucleus were false-colored yellow, red, and blue, respectively. Scale bar: 150 µm.

Figure 8.

Illustration of αSMA staining pattern of the DCP in the DR+ group. (A) A 3D stack was rotated and visualized through the x,z-axis to highlight the connection between the ICP and DCP via the a1 arteriole. (B) Segments of both the ICP and DCP capillaries (Cap); the 3D stack was also viewed through the x,y-axis. The three insets highlight (C) a a1 bifurcation site, (D) an ICP capillary, and (E) a distal segment of DCP capillary. All three capillary segments were found to have αSMA-expressing pericytes (green arrows) and endothelial cells (magenta arrows). In addition, the capillary at the a1 bifurcation was found to have an enlarged diameter (11.5 µm; red asterisk). αSMA, F-actin, and nucleus were false-colored yellow, red, and blue, respectively. Scale bars: 30 µm.

Figure 9.

Schematic representation of αSMA distribution and changes in the development of diabetic retinopathy. Insets provide magnified views of regions of interest. (A) In the control group, αSMA (yellow dots) was predominantly localized within arteries, arterioles, and capillaries on the arterial aspect of the circulation (red). For the venous aspect (blue), αSMA was localized to major venular junctions only. Within the connecting arterioles (a1) between the ICP and DCP, αSMA expression was found to terminate abruptly prior to reaching the DCP (inset I; black arrow). The locations of each vascular plexus are indicated in the retinal layers panel. (B) The DR– group showed additional αSMA expression along veins, venules, and capillaries on the venous aspect of the circulation. Compared to the control group, there was more αSMA expression at venular junctions. In addition, αSMA expression along the a1 arteriole spanned its entire course and extended into the DCP (inset II; black arrow). The DR+ group was characterized by microaneurysms and capillary dropout within the DCP (asterisks; inset III). Compared to the control and DR– groups, there was significantly more αSMA expression along veins, venules, and capillaries on the venous aspect. Within the DCP, αSMA expression was found distal to the a1 arterioles and at venular junctions (inset III; blue arrow).