Early thrombus formation is required for eccentric and heterogeneous neointimal hyperplasia under disturbed flow

Abstract

Background: Anticoagulation and antiplatelet therapy effectively inhibit neointimal hyperplasia (NIH) in both arterial and venous systems but not in arteriovenous fistulae (AVF). The main site of AVF failure is the juxta-anastomotic area that is characterized by disturbed flow compared with laminar flow in the arterial inflow and the venous outflow.

Objectives: We hypothesized that early thrombus formation is required for eccentric and heterogeneous NIH in the presence of disturbed flow.

Methods: Needle puncture and sutured AVF were created in C57BL/6 mice, in PF4-Cre × mT/mG reporter mice, and in Wistar rats. Human AVF samples were second-stage basilic vein transpositions. The tissues were examined by histology, immunofluorescence, immunohistochemistry, and en face staining.

Results: In the presence of disturbed flow, both mouse and human AVF showed eccentric and heterogeneous NIH. Maladapted vein wall was characterized by eccentric and heterogeneous neointima that was composed of a different abundance of thrombus and smooth muscle cells. PF4-cre × mT/mG reporter mice AVF showed that green fluorescent protein-labeled platelets deposit on the wall directly facing the fistula exit with endothelial cell loss and continue to accumulate in the presence of disturbed flow. Neither disturbed flow with limited endothelial cell loss nor nondisturbed flow induced heterogeneous neointima in different animal models.

Conclusion: Early thrombus contributes to late heterogeneous NIH in the presence of disturbed flow. Disturbed flow, large area of endothelial cell loss, and thrombus formation are critical to form eccentric and heterogeneous NIH. Categorization of adapted or maladapted walls may be helpful for therapy targeting heterogeneous NIH.

Keywords: arteriovenous fistula; disturbed flow; neointima; platelet; thrombus.

Figure 1:. Eccentric and heterogenous neointima in the mouse and human arteriovenous fistula (AVF).

A) Illustration pictures showing the concentric and homogenous, eccentric and heterogenous neointimas under laminar flow and disturbed flow. B) Diagram showing the AVF created by puncturing from the aorta to inferior vena cava (IVC) using a 25-gauge needle, black dashed line square showing the juxta-anastomotic area (JAA). Ultrasound measurement of the diameters and waveforms of the aorta and IVC at day 21 after AVF creation at the JAA; A, aorta; IVC, inferior vena cava. C) Mouse AVF sections stained with Verhoeff Van Gieson (EVG) showing the eccentric and heterogenous neointima at day 7 and day 21, black “*” showing the eccentric and heterogenous neointima, black arrow area showing the eccentric and heterogenous neointima on the vein wall; scale bar, 1mm, n=3. D) Illustration picture showing the adapted and maladapted vein wall without or with eccentric and heterogeneous neointima in mouse AVF. E) Low power immunofluorescence photographs showing sections stained with PCNA (green), α-actin (red) and DAPI (blue) at day 7 and 21; white “*” showing the eccentric and heterogenous neointima, white arrow area showing the eccentric and heterogenous neointima on the vein wall; A, aorta; scale bar, 1mm; n=3. F) Light microscope showing the eccentric and heterogenous neointima in human AVF, read line area showing the eccentric and heterogenous neointima, blue line area showing the media, yellow line area showing the adventitia; the maladapted vein wall was categorized into zone 1 and 2 based on the irregular shape; scale bar, 2mm. G) Illustration picture showing the adapted and maladapted vein wall without or with eccentric and heterogeneous neointima in human AVF. H) Immunofluorescence photographs showing human AVF sections stained with CD31 (green), PCNA (green), α-actin (red) and DAPI (blue); white arrow area showing the thrombus residuals in the eccentric and heterogenous neointima on the maladapted vein wall; scale bar, 400μm.

Figure 2:. Anastomotic thrombus transforms to heterogenous anastomotic neointima.

A) Illustration picture showing the anastomotic neointima. B) Temporal sections stained with EVG showing the anastomotic thrombus transformed to neointima in male and female mice without or with CKD, black arrow showing the transformation process from thrombus to neointima; n=3–6. C) Immunofluorescence photographs showing CD31 (red), α-actin (green) and DAPI (blue) at 6 hours, day 1, 7 and 21; yellow arrow showing the transformation process from thrombus to neointima; red arrow showing the blood flow from aorta to IVC; scale bar, 200μm; n=3. D) Anastomotic neointima stained with EVG; and CD31 (red), α-actin (green) and DAPI (blue); yellow arrow showing the thrombus residue. scale bar, 200μm; n=3. E) High power immunofluorescence photographs stained with PCNA (green), α-actin (red) and DAPI (blue) of the heterogenous anastomotic neointima at day 7 and day 21; white dashed line demarcated the SMC-rich and SMC-poor neointima; L, lumen; C, fistula channel; scale bar, 200μm; n=3. F) Bar graphs showing α-actin positive cells area (*, day 7, p<0.0001; day 21, p=0.0178; t-test), α-actin and PCNA dual positive cells (*, p= 0.5729; p=0.0153; t-test) in the SMC-rich and SMC-poor neointima; n=3.

Figure 3:. Eccentric and heterogenous neointima correlates with the maladapted vein wall.

A) Temporal sections stained with EVG showing the adapted and maladapted wall in female and male mice, without or with CKD; n=3–6. B) High power immunofluorescence photographs stained with CD31 (green), α-actin (red) and DAPI (blue); PCNA (green), α-actin (red) and DAPI (blue) of the heterogenous neointima at day 7 and 21; yellow arrows showing the thrombus; scale bar, 200μm, n=3. C) Bar graphs showing endothelial cell coverage; *, day 7, p<0.0001; day 21, p= 0.0010; t-test; n=3. D) Bar graphs showing PCNA and α-actin dual positive cells area; *, day 7, p=0.0191; day 21, p=0.0033; t-test; n=3. E) High power immunofluorescence photographs stained with CD31 (green), tissue factor (TF, red); α-actin (green), collagen-1 (red) and DAPI (blue) of the heterogenous neointima at day 21; yellow arrow showing the TF positive cells inside the heterogenous neointima; scale bar, 200μm, n=3.

Figure 4:. Eccentric and heterogenous neointima locates at the area without endothelial cell coverage under disturbed flow.

A) Sections stained with EVG showing the eccentric and heterogenous neointima (or thrombus at day 3) directly facing the fistula flow exit at day 3 and 7, black arrow showing the fistula flow exit, scale bar, 1mm, n=3. B) Longitudinal section stained with EVG, and stained with vWF (red) and DAPI (blue) showing the fistula anastomosis and fistula exit at day 21, black or white “*” showing the anastomotic neointima; scale bar, 1mm; n=3. C) Sections stained with EVG, and stained with CD31 (green) and DAPI (blue) showing the eccentric and heterogenous neointima directly facing the fistula exit at day 21; black and white arrows show the fistula flow exit, yellow arrow shows the eccentric and heterogenous neointima, red arrow shows the anastomosis, black and white “*” show the anastomotic neointima; scale bar, 1mm; n=3. D) En face stained with CD31 (red) and DAPI (blue) showing different endothelial cell morphology on the vein wall and anastomotic heterogenous neointima under disturbed flow at day 7. Dashed line oval showing the eccentric and heterogenous neointima on the vein wall, note there was no CD31 positive cell coverage on the eccentric and heterogenous neointima surface at day 7; scale bar, 1mm or 100μm n=3.

Figure 5:. Continuous deposition of platelets and thrombus formation are critical for formation of eccentric and heterogenous NIH under disturbed flow.

A) Sections stained with EVG showing the eccentric and heterogenous neointima directly facing the fistula exit at day 14; black arrow showing the fistula flow exit; scale bar, 1mm; n=3. B) Representative images of sections stained with SM22α (red) and DAPI (blue) at the fistula exit level from PF4-mT/mG mice at day 7 and 21; white arrow shows the fistula flow exit; white dashed line circle shows the heterogenous anastomotic neointima; note the SM22α-positive cells encapsulated the green platelets at day 21 but not at day 7; Ao, aorta; scale bars: 1mm. C) Bar graph showing the SM22 α positive cell area in the anastomotic neointima at day 7 and 21; p=0.0005, t-test; n=3. D) Representative low and high power images of sections at the anastomosis and fistula exit levels from PF4-mT/mG mice at day 7; C, fistula channel; yellow arrows show a layer of green platelets deposited on the wall directly facing the fistula exit; scale bars: 400μm or 100μm; n=4 E) Bar graph showing the maladapted and adapted wall length at day 7; p<0.0001, t-test; n=3.. F) Representative low and high power images of sections stained with SM22α (red) and DAPI (blue) at the anastomosis level and fistula exit level from PF4-mT/mG mice at day 21; C, fistula channel; yellow arrows showing a layer of green platelets deposited on the wall directly facing the fistula exit; scale bars: 400μm or 100μm; n=4. G) Bar graph showing the heterogenous neointimal thickness at the level of the anastomosis and the fistula exit at day 21; p=0.0010, t-test; n=3.

Figure 6:. Cannulation of the outflow vein induces eccentric and heterogenous neointima in both human and mouse AVF.

A) Illustration photos showing the cannulation of human and mouse AVF. B) Section stained with EVG showing the eccentric and heterogenous neointima with or without cannulation, irregular dashed line area showing the eccentric and heterogenous neointima; black arrows show the needle cannulation sites. C) Sections stained with EVG showing the heterogenous neointima with or without cannulation, irregular dashed line area showing the cannulation route; scale bar, 1mm or 200μm, n=3. D) Immunofluorescence photographs showing sections stained with CD31 (red), α-actin (green) and DAPI (blue) at day 21 with or without cannulation; 1, 2, 3 showing the different areas; scale bar, 1mm or 200μm; n=3.

Figure 7:. JAA eccentric and heterogenous neointima is universal in sutured anastomosis AVF.

A) Illustration and surgical photos showing the male mouse carotid artery-jugular vein (CA-JV) fistula (end to side) model, ruler marks 1mm. B) Illustration photo and sections stained with EVG showing the JAA at day 7, note the eccentric and heterogenous neointima in the high-power photographs; scale bar, 1mm or 100μm, n=3. C) Immunohistochemistry photographs of the anastomosis and outflow vein (in the JAA) in a male rat JV-CA fistula (end to side) model at day 7, 21 and 42, low and high-power photographs showing the anastomosis and outflow vein stained with α-actin; note the eccentric and heterogenous neointima in the outflow vein; scale bar, 400μm or 100μm, n=3.

Figure 8:. Disturbed flow with limited endothelial cell loss induces homogenous neointima in the AVF inflow artery.

A) Temporal low and high-power photographs stained with EVG showing the gradual changes from thrombus to AVFIA wall from day 0 to day 42 in male mouse; blue arrow showing the thrombus, yellow arrow showing the AVFIA wall, black arrowhead showing the new formed elastin fibers at day 42; IVC, inferior vena cava; scale bar, 400 μm and 200 μm; n=6–10. B) Bar graph showing the thrombus area at day 0, 10 seconds, 6 and 24 hours; p=0.0002, one-way ANOVA; n=3. C) Bar graph showing the luminal area at day 0, 7, 21 and 42; p<0.0001, one-way ANOVA; n=3. D) Illustration photos showing the formation of homogenous neointima of the AVFIA. E) In situ photo showing the AVFIA (yellow dashed line square) at day 21, ruler marks 1mm. F) Series cross-sectional photographs stained with EVG showing homogenous neointima of the AVFIA from cranial to caudal at day 21, black dashed line rectangle showing the AVFIA; black arrow showing the aortic wall, yellow arrow showing the homogenous neointima of the AVFIA; IVC, inferior vena cava; scale bar, 400 μm or 200 μm; n=3. G) Infrequent sections showing AVFIA at day 21, black dashed line square showing the AVFIA, black arrow showing the AVFIA lumen, IVC, inferior vena cava; scale bar, 400 μm or 200 μm. H) Series sections of merged immunofluorescence stained for CD31 (green) and DAPI (blue) of the AVFIA at day 21; from caudal to cephalic (1–4), note the coverage of CD31 positive cells on the AVFIA neointima, white arrow showing the process of diminishing of the native aortic wall dividing the original aorta and AVFIA; scale bar, 200 μm; n=3. I) Representative images of AVFIA from PF4-mT/mG mice at day 7, scale bar, 100 μm; n=5. J) Merged immunofluorescence stained for CD31 (green), α-actin (red), and DAPI (blue); SM22α (green), collagen-1 (red) and DAPI (blue) of the AVFIA at day 21; white dashed line square showing the AVFIA; note the homogenous AVFIA neointima; scale bar, 200 μm; n=3. K) Longitudinal section stained with EVG showing the AVFIA at day 21, black arrow showing the aortic wall, yellow arrow showing the AVFIA; IVC, inferior vena cava; scale bar, 1mm and 400 μm; n=3. L) Longitudinal sections stained for CD31 (green), α-actin (red), and DAPI (blue) of the AVFIA at day 21; white dashed line square showing the AVFIA, white arrow showing the aortic wall, yellow arrow showing the AVFIA; IVC, inferior vena cava; scale bar, 1mm or 200 μm; n=3.