Temporal regulation of venous extracellular matrix components during arteriovenous fistula maturation

Abstract

Purpose: The venous limb of arteriovenous fistulae (AVF) adapts to the arterial environment by dilation and wall thickening; however, the temporal regulation of the expression of extracellular matrix (ECM) components in the venous limb of the maturing AVF has not been well characterized. We used a murine model of AVF maturation that recapitulates human AVF maturation to determine the temporal pattern of expression of these ECM components.

Methods: Aortocaval fistulae were created in C57BL/6J mice and the venous limb was analyzed on postoperative days 1, 3, 7, 21, and 42. A gene microarray analysis was performed on day 7; results were confirmed by qPCR, histology, and immunohistochemistry. Proteases, protease inhibitors, collagens, glycoproteins, and other non-collagenous proteins were characterized.

Results: The maturing AVF has increased expression of many ECM components, including increased collagen and elastin. Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase 1 (TIMP1) showed increased mRNA and protein expression during the first 7 days of maturation. Increased collagen and elastin expression was also significant at day 7. Expression of structural proteins was increased later during AVF maturation. Osteopontin (OPN) expression was increased at day 1 and sustained during AVF maturation.

Conclusions: During AVF maturation, there is significantly increased expression of ECM components, each of which shows distinct temporal patterns during AVF maturation. Increased expression of regulatory proteins such as MMP and TIMP precedes increased expression of structural proteins such as collagen and elastin, potentially mediating a controlled pattern of ECM degradation and vessel remodeling without structural failure.

Figure 1

Histology of AVF maturation. A) Representative photomicrographs stained with hematoxylin and eosin (H&E), days 0–42. Scale bar, 40µm. B) Representative photomicrographs stained with trichrome stain, days 0–42. Scale bar, 40µm. C) Representative photomicrographs stained with van Geison stain, days 0–42. Yellow arrows indicate positive elastin staining. Scale bar, 40µm. D) Bar graph showing wall thickness of venous limb of AVF, days 1–42. n=3. P = .0001. E) Bar graph showing mean number of nuclei per high power field, normalized to day 0. n=3. P = <.0001. F) Bar graph showing relative quantification of collagen staining, normalized to day 0. n=3. P = .0036. G) Bar graph showing relative quantification of elastin staining, normalized to day 0. n=3. P = .002. A, Artery; V, Vein (non-fistula); FA, Arterial limb of AVF; FV, Venous limb of AVF. A.U., arbitrary units

Figure 1

Histology of AVF maturation. A) Representative photomicrographs stained with hematoxylin and eosin (H&E), days 0–42. Scale bar, 40µm. B) Representative photomicrographs stained with trichrome stain, days 0–42. Scale bar, 40µm. C) Representative photomicrographs stained with van Geison stain, days 0–42. Yellow arrows indicate positive elastin staining. Scale bar, 40µm. D) Bar graph showing wall thickness of venous limb of AVF, days 1–42. n=3. P = .0001. E) Bar graph showing mean number of nuclei per high power field, normalized to day 0. n=3. P = <.0001. F) Bar graph showing relative quantification of collagen staining, normalized to day 0. n=3. P = .0036. G) Bar graph showing relative quantification of elastin staining, normalized to day 0. n=3. P = .002. A, Artery; V, Vein (non-fistula); FA, Arterial limb of AVF; FV, Venous limb of AVF. A.U., arbitrary units

Figure 2

Matrix metalloproteinase 2 (MMP-2) expression during AVF maturation. A) Bar graph showing mRNA transcripts of MMP-2 in the AVF venous limb relative to sham vein (normalized to day 0; all samples normalized to GAPDH), days 0–42. n=8. P = <.0001. B) Bar graph showing relative quantification of positive immunostaining of MMP-2 protein in the AVF venous limb (normalized to sham vein), day 7. n=3. P = .0006. C) Representative photomicrograph of immunohistochemistry staining of MMP-2 protein in AVF venous limb at day 7. Scale bar, 40µm. D) Representative photomicrograph of immunohistochemistry staining of MMP-2 protein in sham vein, day 7. Scale bar, 40µm. A, Artery; V, Vein (non-fistula); FA, Arterial limb of AVF; FV, Venous limb of AVF

Figure 3

Matrix metalloproteinase 9 (MMP-9) expression during AVF maturation. A) Bar graph showing mRNA transcripts of MMP-9 in the AVF venous limb relative to sham vein (normalized to day 0; all samples normalized to GAPDH), days 0–42. n=8. P = <.0001. B) Bar graph showing relative quantification of positive immunostaining of MMP-9 protein in the AVF venous limb (normalized to sham vein), day 1. n=3. P = .21 C) Representative photomicrograph of immunohistochemistry staining of MMP-9 protein in AVF venous limb, day 1. Scale bar, 40µm. D) Representative photomicrograph of immunohistochemistry staining of MMP-9 protein in sham vein, day 1. Scale bar, 40µm. A, Artery; V, Vein (non-fistula); FA, Arterial limb of AVF; FV, Venous limb of AVF

Figure 4

Tissue inhibitor of metalloproteinase 1 (TIMP1) expression during AVF maturation. A) Bar graph showing mRNA transcripts of TIMP1 in the AVF venous limb relative to sham vein (normalized to day 0; all samples normalized to GAPDH), days 0–42. n=8. P = <.0001. B) Bar graph showing relative quantification of positive immunostaining of TIMP1 protein in the AVF venous limb (normalized to sham vein), day 1. n=3. P = .002. C) Representative photomicrograph of immunohistochemistry staining of TIMP1 protein in AVF venous limb, day 1. Scale bar, 40µm. D) Representative photomicrograph of immunohistochemistry staining of TIMP1 protein in sham vein, day 1. Scale bar, 40µm. A, Artery; V, Vein (non-fistula); FA, Arterial limb of AVF; FV, Venous limb of AVF

Figure 5

Collagen I (COL I) expression during AVF maturation. A) Bar graph showing mRNA transcripts of COL I in the AVF venous limb relative to sham vein (normalized to day 0; all samples normalized to GAPDH), days 0–42. n=8. P = <.0001. B) Bar graph showing relative quantification of positive immunostaining of COL I protein in the AVF venous limb (normalized to sham vein), day 1. n=3. P = .003. C) Representative photomicrograph of immunohistochemistry staining of COL I protein in AVF venous limb, day 1. Scale bar, 40µm. D) Representative photomicrograph of immunohistochemistry staining of COL I protein in sham vein, day 1. Scale bar, 40µm. A, Artery; V, Vein (non-fistula); FA, Arterial limb of AVF; FV, Venous limb of AVF

Figure 6

Collagen III (COL III) expression during AVF maturation. A) Bar graph showing mRNA transcripts of COL III in the AVF venous limb relative to sham vein (normalized to day 0; all samples normalized to GAPDH), days 0–42. n=8. P = <.0001. B) Bar graph showing relative quantification of positive immunostaining of COL III protein in the AVF venous limb (normalized to sham vein), day 1. n=3. P = .01. C) Representative photomicrograph of immunohistochemistry staining of COL III protein in AVF venous limb, day 1. Scale bar, 40µm. D) Representative photomicrograph of immunohistochemistry staining of COL III protein in sham vein, day 1. Scale bar, 40µm. A, Artery; V, Vein (non-fistula); FA, Arterial limb of AVF; FV, Venous limb of AVF

Figure 7

Osteopontin (OPN) expression during AVF maturation. A) Bar graph showing mRNA transcripts of OPN in the AVF venous limb relative to sham vein (normalized to day 0; all samples normalized to GAPDH), days 0–42. n=8. P = <.0001. B) Bar graph showing relative quantification of positive immunostaining of OPN protein in the AVF venous limb (normalized to sham vein), day 1. n=3. P = .02. C) Representative photomicrograph of immunohistochemistry staining of OPN protein in AVF venous limb, day 1. Scale bar, 40µm. D) Representative photomicrograph of immunohistochemistry staining of OPN protein in sham vein, day 1. Scale bar, 40µm. A, Artery; V, Vein (non-fistula); FA, Arterial limb of AVF; FV, Venous limb of AVF

Figure 8

Fibronectin (Fn) expression during AVF maturation. A) Bar graph showing mRNA transcripts of Fn in the AVF venous limb relative to sham vein (normalized to day 0; all samples normalized to GAPDH), days 0–42. n=8. P = <.0001. B) Bar graph showing relative quantification of positive immunostaining of Fn protein in the AVF venous limb (normalized to sham vein), day 1. n=3. P = .004. C) Representative photomicrograph of immunohistochemistry staining of Fn protein in AVF venous limb, day 1. Scale bar, 40µm. D) Representative photomicrograph of immunohistochemistry staining of Fn protein in sham vein, day 1. Scale bar, 40µm. A, Artery; V, Vein (non-fistula); FA, Arterial limb of AVF; FV, Venous limb of AVF

Figure 9

Cartoon depicting the phases of the ECM changes during the adaptive process of AVF maturation. There is an early phase of ECM breakdown from MMP degradation. A transition phase follows with collagens and elastin reorganization the venous scaffolding. Finally, rebuilding of the matrix of the venous limb of the AVF with larger non-collagenous and glycoproteins. TIMP1 and OPN are highly expressed throughout AVF maturation suggesting regulatory roles of these proteins.