Quantitative analysis of laboratory animals 
Twenty-four rats were included and all were involved in the result analysis, with no deaths or infections. 

Angiography
In adult sham rat hind limbs, few angiographically visible preexisting collaterals were noticed. After 7 days of femoral artery ligation, the number of collateral vessels in the adult rat hind limb was increased, compared with adult sham rat hind limb (P < 0.001). In aged sham rat hind limbs, fewer angiographically visible preexisting collaterals were noticed than in adult sham rat hind limbs (P < 0.05). After 7 days of femoral artery ligation, the number of collateral vessels in the aged rat hind limb was significantly increased, compared with the aged sham rat hind limb (P < 0.001), but less than the adult rat hind limb after femoral artery ligation (P < 0.01; Figure 1).




Detection of cell proliferation 
Ki67 antibody was used as the marker of cell proliferation in this study. Cell proliferation was rarely detected in collateral vessel of sham operation hind limbs. After 7 days of femoral artery ligation, cell proliferation in collateral vessels was active in both adult and aged rats, and the former was higher than the latter: the proliferation index was (13.4±0.5)% vs. (8.4±0.5)% (P < 0.01; Figure 2).




Expression of eNOS and MMP2 in collateral vessels
Our data showed that positive eNOS staining was exclusively localized in endothelial cells. In the sham groups, the expression of eNOS in the arteriole of rat hind limbs was moderate in adult rats and was at a low level in aged rats (P < 0.001). After 7 days of femoral artery ligation, the expression of eNOS in collateral vessels of the adult rat hindlimb was significantly increased, compared with the adult sham rat hind limb (P < 0.001). In aged ligation rat hind limb, the expression of eNOS in collateral vessel was significantly increased, compared with the aged sham rat hind limb (P < 0.001), but lower than adult ligation (P < 0.05; Figure 3).




In arteriole of sham rat hindlimb, expression of MMP2 was very low in adult rat and was moderate in aged rat (P < 0.01). After 7 days of femoral artery ligation, expression of MMP2 in the collateral vessel of the adult rat hindlimb was significantly increased, compared with adult sham rat hind limb (P < 0.001). In the aged ligation rat hind limb, the expression of eNOS in the collateral vessel was significantly increased, compared with the aged sham rat hind limb (P < 0.01), but lower than the adult ligation rat hind limb (P < 0.001; Figure 4).

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Shear stress-induced mir-143-3p in collateral arteries contributes to outward vessel growth by targeting collagen V-α2. Arterioscler Thromb Vasc Biol. 2020;40(5):e126-e137. [13] HOLLANDER MR, JANSEN MF, LHGA H, et al. Stimulation of collateral vessel growth by inhibition of galectin 2 in mice using a single-domain llama-derived antibody. J Am Heart Assoc. 2019;8(20):e012806.  [14] HIRSCH AT. Critical limb ischemia and stem cell research: anchoring hope with informed adverse event reporting. Circulation. 2006;114(24): 2581-2583. [15] ROSENZWEIG A. Cardiac cell therapy--mixed results from mixed cells. N Engl J Med. 2006;355(12):1274-1277. [16] YINJUAN T, JIANJUN W, YINGLU G, et al. Effect of atorvastatin on LOX-1 and eNOS expression in collateral vessels of hypercholesterolemic rats. Nan Fang Yi Ke Da Xue Xue Bao. 2019;39(11):1265-1272. [17] SUN N, NING B, BRUCE AC, et al. In vivo imaging of hemodynamic redistribution and arteriogenesis across microvascular network. Microcirculation. 2020;27(3):e12598. [18] FADINI GP, SPINETTI G, SANTOPAOLO M, et al. Impaired regeneration contributes to poor outcomes in diabetic peripheral artery disease. Arterioscler Thromb Vasc Biol. 2020;40(1):34-44.  [19] FUJITA K, TANAKA K, YAMAGAMI H, et al. Detrimental effect of chronic hypertension on leptomeningeal collateral flow in acute ischemic stroke. Stroke. 2019;50(7):1751-1757. [20] ZHANG H, JIN B, FABER JE. Mouse models of Alzheimer’s disease cause rarefaction of pial collaterals and increased severity of ischemic stroke. Angiogenesis. 2019;22(2):263-279. [21] YANG BL, WU S, WU X, et al. Effect of shunting of collateral flow into the venous system on arteriogenesis and angiogenesis in rabbit hind limb. Acta Histochem Cytochem. 2013;46(1):1-10. [22] LUO MY, YANG BL, YE F, et al. Collateral vessel growth induced by femoral artery ligature is impaired by denervation. Mol Cell Biochem. 2011;354(1-2):219-229. [23] ITO WD, ARRAS M, WINKLER B, et al. Monocyte chemotactic protein-1 increases collateral and peripheral conductance after femoral artery occlusion. Circ Res. 1997;80(6):829-837.  [24] RIVARD A, FABRE JE, SILVER M, et al. Age-dependent impairment of angiogenesis. Circulation. 1999;99(1):111-120.  [25] WESTVIK TS, FITZGERALD TN, MUTO A, et al. Limb ischemia after iliac ligation in aged mice stimulates angiogenesis without arteriogenesis. J Vasc Surg. 2009;49(2):464-473. [26] LI Y, ZHENG J, BIRD IM, et al. Effects of pulsatile shear stress on nitric oxide production and endothelial cell nitric oxide synthase expression by ovine fetoplacental artery endothelial cells. Biol Reprod. 2003;69(3):1053-1059.  [27] DAI X, FABER JE. Endothelial nitric oxide synthase deficiency causes collateral vessel rarefaction and impairs activation of a cell cycle gene network during arteriogenesis. Circ Res. 2010;106(12):1870-1881.  [28] RAFFETTO JD, KHALIL RA. Matrix metalloproteinases and their inhibitors in vascular remodeling and vascular disease. Biochem Pharmacol. 2008;75(2):346-359.  [29] CHENG XW, KUZUYA M, NAKAMURA K, et al. Mechanisms underlying the impairment of ischemia-induced neovascularization in matrix metalloproteinase 2-deficient mice. Circ Res. 2007;100(6):904-913.  [30] HAAS TL, DOYLE JL, DISTASI MR, et al. Involvement of MMPs in the outward remodeling of collateral mesenteric arteries. Am J Physiol Heart Circ Physiol. 2007;293(4): H2429-H2437.

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Ischemic disease (such as peripheral arterial occlusion) is one of cardiovascular diseases. The incidence of peripheral arterial disease (PAD) increases with age. The incidence of PAD is 5%-10%, while it is 15%-20% in their 70s[1]. In addition, the 
mortality of ischemic cardiovascular disease is also higher in the elderly population[2]. With the increasing trend of aging, more people will suffer from PAD. Although clinical surgical angioplasty, such as percutaneous transluminal angioplasty [3-4], can recanalize obstructed vessels, but some patients have postoperative restenosis. Moreover, some patients are not suitable for surgery or endovascular treatment [5]. Therefore, there is an urgent need to find new treatment strategies for ischemic diseases. 

As early as the 1950s, the British cardiologist Fulton observed that when some patients with severe coronary atherosclerosis died, the cause of death was not due to cardiac ischemia[6]. Fulton and others performed angiography on the heart of these patients after death. Another study indicated that patients with long-term severe blockage of coronary arteries often have extensive collateral vessel growth[7]. These studies show that when arterial blood vessels are blocked, small arterial connections that originally exist between the main arterial branches but do not have the function of transporting blood can develop into larger collateral vessels with blood-conducting function, and then replace the blocked arteries to supply ischemia tissue. The mature collateral vessels can partially or completely restore blood flow in the ischemic area. Therefore, scientists believe that promoting collateral vessel growth is an ideal strategy for the treatment of ischemic diseases[8]. Because of the importance of collateral vessels to ischemic tissues and organs, the study of collateral vessel growth mechanisms has become a hot spot in the field of prevention and treatment of ischemic diseases. A large number of studies have shown that the mechanism of collateral vessels growth mainly includes three aspects: fluid shear stress, inflammation and growth factors[9]. 

Animal experiments have found that injection of angiogenic cells, pigment epithelium-derived factor[10], capsaicin[11], miR-143-3p[12], single-domain llama-derived antibody[13], bone marrow-derived monocytes or growth factors into experimental animals significantly promotes the growth of collateral vessels. However, a large number of clinical trials have been ineffective[14-15]. Comparing the significant effects of animal experiments and disappointing clinical trial results, we found that many factors may affect the growth of collateral vessels in clinical trials, such as hypercholesterolemia[16], obesity[17], diabetics [18], chronic hypertension[19], Alzheimer’s disease[20] and age. In most laboratories, young animals are used, and most of the patients with ischemic diseases are elderly, indicating that aging may be an important risk factor affecting the growth of collateral vessels. Therefore, this study intends to explore the effect of ageing on the growth of collateral vessels in ischemic hind limbs in adult and elderly rat models of femoral artery ligation. 
中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Design
This was an animal experiment.

Time and settings
The experiment was completed in the Histology and Embryology Laboratory of Central South University from September 2016 to December 2019.

Materials
Purified mouse anti-endothelial nitric oxide synthase (eNOS)/nitric oxide synthase (NOS) type III (BD Transduction Laboratories, USA), ki-67 rabbit monoclonal antibody, biotinylated anti-mouse IgG (H+L) affinity purified, biotinylated anti-rabbit IgG (H+L) affinity purified and dylight 549 streptavidin (Vector Laboratories, USA), anti-matrix metalloproteinase 2 (MMP2) antibody and anti-α-smooth muscle actin antibody (Alexa Fluor R488) (Abcam, UK).

Methods
Animal models
Sprague-Dawley (SD) rats were divided into adult sham group (n=6), adult ligation group (n=6), aged sham group (n=6), and aged ligation group (n=6). In the ligation groups, femoral artery ligation was performed on the hind limbs of rats. Surgery for femoral artery ligation surgery was as follows: 10% chloral hydrate (0.35 mL/100 g) was used for anesthesia via intraperitoneal injection. The rat was on the supine position and their limbs were fixed on the operating table, followed by removing the hair in the groin, touching the femoral artery in the groin and making a 2.5 cm incision along the direction of the blood vessel. The skin, fascia and femoral artery were separated layer by layer under the stereo microscope. The femoral artery and femoral vein were carefully separated, and then the femoral artery was double-ligated with a 5-0 surgical suture, and the skin was sutured with 3-0 surgical suture in order. All surgical instruments were sterilized and the operation was performed aseptically. After the operation, the rats were fed in a single cage in the animal room at 22 °C. They were free access to food and water. A total of 24 SD rats (half male and half female) were used for the experiment. 

Preparation of tissue samples
As described in previous studies[21-22], endothelial cells were activated by 1-week fluid shear stress stimulation. Therefore, in the present study, the rats were killed 1 week after operation. The gracilis muscle was isolated from each animal. According to the angiographic results, during the growth of collateral vessels, the gracilis muscle usually contained 2-4 collateral vessels. For immunofluorescence, all samples were stored at -80 °C refrigerator until use.

Postmortem angiography
Postmortem angiography was executed by instilling the contrast agents as previously outlined[23]. Gelatine (40 g) was dissolved in 200 mL of distilled water (45 °C) with consecutively stirring. Subsequently, lead oxide (200 g) was appended and sufficiently combined. Rats were anaesthetized and the aorta was cannulated. Afterwards, the animals were bled and steeped in water (45 °C). Then, the contrast agent was injected until full filling of the distal femoral stump. The rats were straightway laid on broken ice, and the contrast agent was allowed to harden with successive pressure. X-ray images were taken in an X-ray chamber and conveyed to a computer, which were used for identifying and quantifying the collateral vessels. 

Immunofluorescence
Frozen slices were chipped 7 μm in depth and fixed in 4% paraformaldehyde. Afterwards, the slices were incubated in 5% bovine serum albumin followed by incubated with first antibodies (Table 1). Biotinylated second antibodies were followed. Subsequently, the slices were incubated with dylight 549 streptavidin and α-Smooth Muscle-Cy3™. 4’,6-Diamidino-2-phenylindole was used for staining the nuclei. The slices were cover slipped and observed with a confocal microscope. PBS was used instead of the primary antibody as negative control for excluding non-specific response.

Main observation indicators 
Ki67 positive cells were quantified using a confocal microscope, and counted at 40×. The ratio of the number of Ki67 positive nuclei to the total number of vascular wall nuclei was used as the proliferation index. The quantification of immunofluorescence intensity of eNOS and MMP2 was performed using the quantitation software Image Pro-Plus (IPP). The fluorescence intensity was presented as integrated optical density (IOD).
 
Statistical analysis
All data are expressed as the mean± SEM. Statistical comparisons among groups were performed using Student’s t-test. A P < 0.05 was deemed statistically significant.

The results of this study demonstrate that: (1) in aged rats, collateral vessel growth in hindlimb ischemic model was model was impaired, showing the number of collateral vessel less than that in adult rats; (2) compared with the adult ligation group, the aged ligation group showed that the number of collateral vessels was reduced, Ki-67 positive cells in vascular walls were reduced, and the expression levels of eNOS and MMP2 were also decreased.
The femoral artery ligation model in experimental animals is a common model to observe the growth of collateral vessels induced by acute hindlimb ischemia. Scientists used this model to find that hind limb blood flow in aged animals is less than that in adult animals after acute ischemia of hindlimbs, and the recovery of hind limb function is weakened, which is believed to be due to the influence of aging on angiogenesis[24]. Similarly, another acute hindlimb ischemia model induced by ligation of iliac arteries and veins found that the ability of aged rats could still maintain potent angiogenesis to some extent, but the recovery of hindlimb blood flow and function in aged rats is far less than that of adult rats, and aged rats mainly induce angiogenesis, while collateral vessel growth is impaired, indicating that aging leads to defects in arteriogenesis[25]. In this study, we found that the number of collateral vessels was smaller in aged rats than in adult rats at 7 days after femoral artery ligation. This further proves that aging leads to decreased collateral vessel growth capacity.
Nitric oxide and NOS can regulate the function of vascular endothelial cells. Li et al. [26] found that fluid shear stress increased nitric oxide production by increasing the activity of eNOS. Dai et al. [27] found that compared with wild-type mice, eNOS knockout mice showed blood flow and more severe shrinkage injury after 3 weeks of femoral artery ligation, which was caused by reduced collateral vessel remodeling. This indicates that the growth of collateral vessels due to shear stress depends on the expression of eNOS. It was found in the experiment that the expression of eNOS in the collateral vessels of aged rats was significantly lower than that of adult rats in the sham groups and the ligation groups. Therefore, the low expression of eNOS in aged rats may affect the growth of collateral vessels.

MMP2 is a group of zinc-containing enzymes that are involved in the degradation of the extracellular matrix[28]. In normal arterioles, smooth muscle cells are surrounded by a basement membrane, which restricts the migration and proliferation of smooth muscle cells; the inner elastic membrane between the intimal and medial membranes also limits smooth muscle cell migration. MMP2 can degrade the basement membrane and elastic membrane.
CHENG et al. [29] applied hind limb ischemia models with MMP2 +/+ and MMP2-/-mice and found that there were less developed collateral vessels in MMP2-/-mice. This shows that the loss of MMP2 impairs the growth of collateral vessels. This is consistent with the findings of Haas et al.[30] that MMP2 promotes the growth of collateral vessels. In the present study, we found that after ligation of the femoral artery, the expression of MMP2 in the collateral vessels of adult rats was significantly enhanced, causing the degradation of elastic components and extracellular matrix, which was beneficial to the outward growth of collateral vessels. However, the expression of MMP2 was also increased in aged rats after the same operation, but not as obvious as in adult rats.
In this present study, we found that the expression of eNOS and MMP2 in the collateral vessel of the aged ligation group was lower than that in the adult ligation group. Therefore, we speculate that the low expression of eNOS and MMP2 during the growth of collateral vessels may be an important mechanism for the damage of the growth of the collateral vessels in the aged rats.
In conclusion, our data demonstrate that aging impairs the growth of collateral vessels in the rat model of hind limb ischemia. The low expression of eNOS and MMP2 in the collateral vessels may be an important mechanism of impaired collateral vessel growth. However, we should continue to study the mechanism by which aging leads to impaired growth of collateral vessels, thereby providing a theoretical basis for the treatment of senile ischemic diseases.
中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

文题释义：
侧支循环：为血管主干近侧分支和远侧分支之间所形成的血管网。
代偿性循环：机体某一局部的主要血管(动脉或静脉)的血流受阻后，该部原有吻合支的血管扩张，形成旁路，使血液迂回地通过这些旁路，恢复了循环，这种循环途径称为侧支循环，又称为代偿性循环。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

动物实验发现，将血管生成细胞或生长因子注入实验动物可显著促进侧支血管的生长。然而，临床随机对照试验包括给予重症下肢缺血或心肌梗塞患者血管生成细胞显示没有改善缺血组织血流恢复。#br#

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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