Relationship of PIEZO1 and PIEZO2 vascular expression with diabetic neuropathy

Abstract

Introduction: Diabetic distal symmetric polyneuropathy (DDSP) is the most prevalent form of diabetic peripheral neuropathy, and 25% of patients develop pain in their toes. DDSP is associated with increased cutaneous microvessel density (MVD), reduced skin blood flow, endothelial dysfunction, and impaired fluid filtration with vasodilation. The Piezo family of mechanosensitive channels is known to be involved in the control of vascular caliber by converting mechanical force into intracellular signals. Furthermore, Piezo2 is particularly involved in peripheral pain mechanisms of DDSP patients. To date, very little is known about the number, structure, and PIEZO expression in cutaneous blood vessels (BVs) of individuals with DDSP and their relation with pain and time span of diabetes. Methods and results: We studied microvessels using endothelial markers (CD34 and CD31) and smooth cell marker (α-SMA) by indirect immunohistochemical assay in sections of the glabrous skin of the toes from patients and controls. MVD was assessed through CD34 and CD31 immunoreaction. MVD determined by CD34 is higher in short-term DDSP patients (less than 15 years of evolution), regardless of pain. However, long-term DDSP patients only had increased BV density in the painful group for CD31. BVs of patients with DDSP showed structural disorganization and loss of shape. The BVs affected by painful DDSP underwent the most dramatic structural changes, showing rupture, leakage, and abundance of material that occluded the BV lumen. Moreover, BVs of DDSP patients displayed a Piezo1 slight immunoreaction, whereas painful DDSP patients showed an increase in Piezo2 immunoreaction. Discussion: These results suggest that alterations in the number, structure, and immunohistochemical profile of specific BVs can explain the vascular impairment associated with painful DDSP, as well as the temporal span of diabetes. Finally, this study points out a possible correlation between increased vascular Piezo2 immunostaining and pain and decreased vascular Piezo1 immunostaining and the development of vasodilation deficiency.

Keywords: DPN; Piezo; blood vessels; diabetic distal symmetrical polyneuropathy; painful and painless; skin.

FIGURE 1

Bar graph showing the microdensity blood vessels/4 mm² defined by CD31 and CD34 in the glabrous toes skin of healthy individuals (n = 10), DDSP-NP ST (n = 5) and LT (n = 5), and DDSP-P ST (n = 5) and LT (n = 5). Comparison of the number of blood vessels positive for CD31 (green) and CD34 (orange) in the dermis (A); comparison of CD31-positive blood vessels in the papillary (blue) vs. reticular (gray) dermis (B); and comparison of CD34-positive blood vessels in the papillary (blue) vs. reticular (gray) dermis (C). ST, short term; LT, long term. I—I is SD.

FIGURE 2

Immunostaining for α-SMA and CD34 in the glabrous toes skin. α-SMA antibody identifies the muscular layer in arteries and venules (A, C–J). Blood vessels of neuropathy patients showed, in some cases, a lack of lumen [black arrow in (F, J)] in the papillary dermis. In the reticular dermis, the blood vessels of healthy controls present a constant muscle layer and with intense immunoreactivity (A), whereas patients with diabetic neuropathy present an irregular immunostaining without a continuous α-SMA immunopositive muscle layer [see red arrows (C, E, G)] and the muscular layer thickness decreased [red arrow in (I)], even blood vessel wall was broken [black arrow in (I)]. CD34 immunostaining marks endothelium and single smooth muscle cells. In healthy individuals (B) and in patients with DDSP NP (K, L, O, P), a continuous and regular endothelium is observed, while patients with DDSP-P present interruptions in the course of the endothelium [red arrow in (Q)]. Black arrows indicate blood vessels in the papillary dermis that present a (M,N,Q,R) closed lumen in DPN patients [see (L, N P, R)]. The green asterisks indicate accumulations of debris in the lumen of the vessels (I, Q).

FIGURE 3

Number of microvessels in the skin of healthy adults (A) and with diabetes neuropathy (B–E). Sections are stained by immunohistochemistry to show the number of blood vessels defined by a CD31, an endothelium marker (A–E). Blood vessels are identified with a red spot in the pictures (A–E). The number of blood vessels is shown in a small red box in the same images. A higher number of blood vessels are noted in patients with neuropathy, especially in DDPS-P (D,E). In the papillary dermis of patients with neuropathy, (F,H,I,J,K) a reduced capillary luminal area [red arrow (H,L,K)] was observed. With respect to the reticular dermis, the immunoreaction with CD31 allows observing a continuous and regular endothelium in the healthy individuals (F,G), while the patients with DDPS (L,M,N,O) present interruptions in the endothelium in some cases [see red arrows (L,O)] or multiple layers [black arrow in (N, O)].

FIGURE 4

Expression of the PIEZO1 ion channel in the glabrous skin of the feet. The immunoreaction in healthy controls is intense in the endothelium of the vessels of the papillary dermis (A) and in the endothelium and muscle layer of the arterioles (B). In patients with neuropathy, there is almost no immunoreaction in the endothelium of papillary vessels [red arrows indicate PIEZO1-negative vessels in (C, E, G, I)]. The muscular layer of the arteries presents diffuse labeling in diabetic patients, as well as a discontinuous endothelium [red arrows indicate loss of endothelial continuity in (D, F, H, J)].

FIGURE 5

Immunoexpression of PIEZO2 in the vessels of the glabrous skin of the feet. In the healthy controls, an immunoreaction is observed in the endothelium of the veins (A), as well as in the muscular layer of the arteries (B). In patients with DDPS-NP, (C,D,G,H) the intensity of the reaction is lower and several blood vessel are negative [red arrows in (C,G)]; Moreover ie cells clot to endothelium are arectuve ub arterues [red arrow in (H)] . Patients with DDPS-P present a more intense immunoreaction in the papillary vessel (E,I), while arteries in the reticular dermis show a diffuse immunoreaction with respect to healthy individuals (F,K,J).

FIGURE 6

Double fluorescence of CD34/α-SMA and PIEZO2 ion channels in blood vessels. Healthy individuals show PIEZO2 expression in the endothelium of blood vessels (A) and arteries (B). Patients with painless diabetic neuropathy show a loss of immunoreaction [white arrow in (C, E, G)]. However, PIEZO2 immunostaining in green is higher in patients with painful neuropathy (D, F, H), being more striking in the endothelium of the vessels of the papillary dermis [see arrow in (D, F)]. Objective ×63/1.40 oil; pinhole 1.37; xy resolution 139.4 nm and Z resolution 235.8 nm.