Role of vascular endothelial progenitor cells in construction of new vascular loop

YMVRE-03338; No. of pages: 11; 4C: Microvascular Research xxx (2013) xxx–xxx

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Role of vascular endothelial progenitor cells in construction of new vascular loop Kuihua Zhan a,b,⁎, Lun Bai b,⁎⁎, Jianmei Xu b a b

School of Mechanical and Electronic Engineering, Soochow University, 178 Ganjiang East Road, Suzhou 215006, China College of Textile and Clothing Engineering, Soochow University, 178 Ganjiang East Road, Suzhou 215006, China

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Article history: Accepted 28 June 2013 Available online xxxx

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Since bone marrow-derived endothelial progenitor cells (EPCs) have been detected in adult peripheral blood, the mode of vasculogenesis in the adult tissue has caught attention in field of vascularization research. To confirm the role of EPCs in construction of new vascular loop, we took the biomaterial scaffold implanted into adult rat as an experimental model to observe and examine the actions of the EPCs in neovascularization of the material by immunohistochemistry and transmission electron microscopy. Additionally, by establishing a chemotactic migration model for vascular endothelial cells (ECs) and EPCs, the migrations of ECs and EPCs were explored in simulations. The results of 20,000 simulations showed that the number of the vascular loops assisted by the EPCs was 2–5 times that of the vascular sprouts being naturally joined. Based on the results of experiments and simulations, we conclude that the EPCs are able to assist the angiogenic sprouts in joining under the condition of plenty of the EPCs being mobilized, which aggregate at sites close to sprout tips, forming a cell cord and differentiating to ECs in situ, and become vessel segments between neighboring sprouts. This suggests that there is a difference between the adult and embryo in the manner of vasculogenesis and that a small number of EPCs can play an important role to make the new blood vessels achieve rapid functionalization. © 2013 Published by Elsevier Inc.

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Introduction

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The various angiogenesis, containing sprouting and nonsprouting angiogenesis, may appear in the adult tissue dominated by physiological and pathological factors (Asahara et al., 1997; Carmeliet, 2000; Djonov et al., 2002, 2003; Gerhardt et al., 2003). Since bone marrow-derived endothelial progenitor cells (EPCs) have been detected in adult peripheral blood (Asahara et al., 1997), the mode of vasculogenesis in the adult tissue has caught attention in the field of vascularization research. Although the researches have indicated that EPCs may integrate into damaged endothelial monolayer to repair injured vessels and may home in on the extravascular tissue to participate in neovascularization of ischemic organs (Hristov et al., 2003), there are only a few studies from the concept of avascular

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Abbreviations: Ang-1, Angiopoietin-1; CD133, 5-transmembrane glycoprotein; Dll4, Delta-like 4 protein; EC, Endothelial cell; EPC, Endothelial progenitor cell; FN, Fibronectin; G-CSF, Granulocyte colony-stimulating factor; GM-CSF, Granulocyte/Macrophage colony-stimulating factor; HIF-1α, Hypoxia-inducible factor 1α; MAPC, Multipotent adult progenitor cell; MMPs, Matrix metalloproteinases; Notch, Transmembrane receptor protein; SDF-1, Stromal cell-derived factor-1; TEM, Transmission electron microscopy; VEGF, Vascular endothelial growth factor; VEGFR, Vascular endothelial growth factor receptor. ⁎ Correspondence to: K. Zhan, School of Mechanical and Electronic Engineering, Soochow University, 178 Ganjiang East Road, Suzhou 215006, China. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (K. Zhan), [email protected] (L. Bai), [email protected] (J. Xu).

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tissue in the adult on the role of EPCs. The avascular tissues, such as avascular solid tumor (Folkman et al., 1971) and early wound healing tissue (Gurtner et al., 2008), are newly formed tissues and possess common characteristics that the tissue cells, such as inflammatory cells, fibroblasts and tumor cells, produce angiogenic factors due to hypoxia or inflammatory reaction (Shweiki et al., 1992; Carmeliet and Jain, 2000; Hillen and Griffioen, 2007; Kerbel, 2008) after they infiltrate into the avascular area. Among those angiogenic factors are vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), etc. (Papapetropoulos et al., 2000; Inoue et al., 2002; Gehling and Ergün, 2008), VEGF is an angiogenic factor with high specificity (Maxwell et al., 1997). The VEGF activates vascular endothelial cells (ECs) on the vascular plexus to wrap around the avascular tissue to trigger angiogenic sprouting (Brat and Mapstone, 2003). It has been determined that sprouting angiogenesis is a main mode of neovascularization (Roca and Adams, 2007) and the contact of the vascular sprouts leads to new vascular loops (Ramjaun and Hodivala-Dilke, 2009; Eilken and Adams, 2010). Furthermore, a great number of experiments have confirmed that the specific cytokines, such as VEGF, SDF-1 (stromal cell-derived factor-1), GM-CSF (granulocyte/ macrophage colony-stimulating factor), G-CSF (granulocyte colonystimulating factor), may mobilize EPCs from bone marrow to peripheral blood (Asahara et al., 1999; Falco et al., 2004; Huang et al., 2004), and the EPCs may migrate to sites of active neovascularization to participate in construction of new blood vessels (Isner and Asahara, 1999). However, a number of questions remain unanswered and are

0026-2862/$ – see front matter © 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.mvr.2013.06.010

Please cite this article as: Zhan, K., et al., Role of vascular endothelial progenitor cells in construction of new vascular loop, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.06.010

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Biomaterial scaffolds for implantation

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The porous silk fibroin film of about 1 mm thickness was prepared by adding certain specific additives to silk fibroin aqueous solution, which was then lyophilized under certain freeze-drying conditions. The average pore size of the material was 100–200 μm and the pores were connected to each other. The material was cut into piece with the size of 2 cm × 1 cm and was treated by irradiation sterilization.

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Materials

Antibodies for immunohistochemistry

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Experimental animals

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SD rats for experiments were all healthy, male, between 200 g and 250 g in weight and were provided by the Experimental Animal

Rabbit anti-rat CD133 antibody (1:50) was purchased from Wuhan Boster Bio-engineering Limited Company in China. Secondary antibody detection kit (k500711) was purchased from Gene Tech (Shanghai) Company Limited in China.

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Center of Soochow University. The experiments were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals and the Management Committee of Experimental Animal Center (Medical College of Soochow University, Suzhou, China).

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worthy of further study, for instance, whether the EPCs participate in construction of new vascular loops and whether the formation of a new vascular loop is totally dependent on the natural anastomosis of vascular sprouts, that is, the contact of sprout tips, when EPCs are mobilized into peripheral blood and exude to an avascular area. It is becoming one of current concerns as to which factors promote the fusion of neighboring tip cells to add new vessel loops (Fantin et al., 2010). In this study, taking the biomaterial scaffold implanted into adult rat as an experimental model for neovascularization of an avascular tissue, we observe the actions of EPCs in the vascularization process from nothing to something by histological and morphological examinations, and simulate the formation of new vascular loops by mathematical modeling for the migrations of ECs and EPCs. The aim is to investigate the role of EPCs in construction of new vascular loops and the difference between the adult and embryo in the manner of vasculogenesis.

Methods

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Animal experiments

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Surgery for implantation The SD rats were anesthetized with 4% chloral hydrate (1 ml/100 g). A piece of full thickness back skin was cut off, which was larger than 2 cm × 1 cm, and partial dermis was removed. The ready porous silk fibroin film was implanted into the wound bed and then the injured flap was put on the material, sutured. The surgery and subsequent feeding were done in the Experimental Animal Center of Soochow University.

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Mathematical modeling

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Chemotactic responses of ECs and EPCs Here we investigate the chemotactic migration of ECs and EPCs, which respond to the concentration gradient of the VEGF as a major angiogenic chemokine (Gerhardt et al., 2003). In an avascular tissue, the sprouting parent vessels lie on one side of the tissue cell population (see Fig. 1), and the cells from the host tissue produce VEGF, which is mediated by hypoxia-inducible factor-1α (HIF-1α). Under normal circumstances, the ECs on capillaries and microvessels are in stationary phase of cell cycle. When the tissue enters the hypoxic or inflammatory state, due to the action of the VEGF, the ECs lose the contact inhibition, leading to an increased vascular permeability (Ferrara et al., 2003), and the activated ECs on the parent vessel produce matrix metalloproteinases (MMPs) which can dissolve the basement membrane and become motile, migrating out of the parent vessel to become tip ECs of the vascular sprouts (Distler et al., 2003). Because the amount of the VEGF is positively correlated with hypoxic intensity (Gerber et al., 1997; Carmeliet and Jain, 2000; Fong, 2008), the tissue cells far from the parent vessel are severely hypoxic and will highly express VEGF; the cells near the parent vessel are slightly hypoxic and will express VEGF to a low degree, and the existing VEGF gradient will steepen due to a protein gradient of VEGFR1, which is produced by ECs on the parent vessel and secreted into the local milieu (Kearney et al., 2004), so the VEGF molecules around the parent vessel form a relatively steady-state concentration gradient to aid the chemotactic migration of the tip ECs on sprouting parent vessel. The growth rate and density of new vascular sprouts depend on the induced intensity of VEGF (Gerhardt et al., 2003; Eilken and Adams, 2010). Once the ECs on the existing blood vessel are activated by high-intensity (high-concentration) VEGF, the ECs will express a large number of receptors

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Histological and morphological examinations The implanted materials were sampled at several time points after implantation and were prepared using two methods namely optical microscopy and transmission electron microscopy (TEM) observations. The sampled materials with the flaps attached were fixed in 10% formalin at room temperature, paraffin-embedded and subsequently made into sections (4 μm) for CD133 immunohistochemical staining. Additionally, the materials were separated from the flaps, sliced carefully into blocks of about 1 mm3 and placed in 2.5% glutaraldehyde solution at 4 °C. The samples were then rinsed in phosphate buffer and post-fixed in 1% osmium tetroxide for an hour, dehydrated in ascending grades of acetone, embedded and made into the ultrathin sections (70–80 nm), stained with uranyl acetate and lead nitrate and viewed with a Hitachi H-600 electron microscopy. The porous silk fibroin film could induce regeneration of rat skin (Altman et al., 2003; Guan et al., 2010). The regenerative tissue is considered as avascular tissue in the early period after implantation, which consists of two parts: (1) the wound bed between the porous silk fibroin film and host muscle tissue, (2) the interior of the material, which has been infiltrated by cells being mainly made of inflammatory cells from the host tissue. The vascular plexus that will wrap around the avascular tissue will be sprouting parent vessels (see Fig. 1). The distributions of CD133 positive cells in the two avascular tissues shown in Fig. 1 were observed under optical microscopy, and the ultrastructures of ECs during angiogenesis in the material were captured by TEM.

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Please cite this article as: Zhan, K., et al., Role of vascular endothelial progenitor cells in construction of new vascular loop, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.06.010

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Migration model of cells Taking a small piece of avascular tissue which contains a horizontal parent blood vessel as the research object, we divide it into several discrete lattices and denote their horizontal and vertical location states by i and j, respectively. The location state j starts from the parent vessel and is in the direction of the VEGF source. The state-space is i ∈ (− ∞, …, − 1, 0, 1, …, ∞), j ∈ [0, 1, 2, …, ∞). Let ξ(t) represent the location of the cell at t time, t ∈ T, T ∈ [0, 1, 2, …, ∞). Supposing that the VEGF source is a line source far from the parent blood vessel and parallel to it, and the direction of VEGF concentration gradient is vertical to the parent vessel, we establish a discrete model and consider that the VEGF concentration near the parent vessel is low and that far from the parent vessel it is high, and the tip ECs migrate in a random directional way to the direction of j increment, consistent with the VEGF concentration gradient. The model stipulates that the movement must take place in the lattices, for instance, after a unit time interval (t → t + 1), the cell moves one lattice point either to the front (location state (i,j) → (i, j + 1)) or the oblique front, left or right

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(VEGFR) and will bind to the surrounding ligands (Gerhardt et al., 2003). Because the vascular sprouts branch out from the parent blood vessel based on ECs' competition for the cell tip position through Dll4/Notch and Jagged1/Notch signaling, there is certain spacing between the sprouts (see Fig. 2 (a)) (Hellström et al., 2007; Roca and Adams, 2007; Benedito et al., 2009; Eilken and Adams, 2010). In this process, the tip cell as the base of the emerging sprout guides growth of the angiogenic sprout along the concentration gradient of VEGF, and the adjacent stalk cells following the tip cell provide new cells for the growing vascular sprout via the VEGF/VEGFR pathway (Gerhardt et al., 2003). The stalk cells express VEGFR, absorbing the surrounding VEGF, proliferating after phosphorylation (Ferrara et al., 2003), so that the VEGF around the vascular sprouts is consumed, resulting in a down-regulation of VEGF concentration, and the concentration between the two sprouts is relatively high (see Fig. 2 (b)). Thus, we may infer that the concentration of the VEGF around vascular sprouts is lower than that between two vascular sprouts, where the relatively high concentrations of VEGF and SDF-1 coordinating with VEGF (Petit et al., 2007) lead the bone marrow-derived EPCs to be mobilized (Hicklin and Ellis, 2005; Arbab et al., 2006) and to migrate out from the parent blood vessel between the two angiogenic sprouts into the tissue, see Fig. 2 (b). In Fig. 2, the staining intensity means concentration of VEGF molecules. Based on the analysis of the chemotactic responses of the tip EC and bone marrow-derived EPCs, we consider that the chemotactic migration of the tip EC determines the growth path of the vascular sprout, and the migration behavior of the EPCs may reflect the distribution of EPCs in the tissue, which is then used to reveal the role of EPCs in the construction of new vascular loops.

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Fig. 1. Avascular tissue in the early period after implanting porous silk fibroin film into adult rat. The avascular tissue consists of two parts: (1) the wound bed between the porous silk fibroin film and host muscle tissue, (2) the interior of material, which has been infiltrated by cells mainly made of inflammatory cells from host tissue.

Fig. 2. VEGF concentration gradient and angiogenic sprouting in avascular tissue. The staining intensity means concentration of VEGF molecules. (a) The activated ECs, termed tip cells, migrate towards VEGF with high concentration, and vascular sprouts are formed. (b) The ECs on the angiogenic sprouts express VEGF receptors, which bind to VEGF, leading to a reduction of the VEGF concentration around the sprouts, while the bone marrow-derived EPCs are mobilized and migrate into the avascular tissue from the parent vessel between the neighboring sprouts, where VEGF concentration is relatively high.

Please cite this article as: Zhan, K., et al., Role of vascular endothelial progenitor cells in construction of new vascular loop, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.06.010

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(location state (i,j) → (i − 1, j + 1) or (i,j) → (i + 1, j + 1)) (see Fig. 3, arrows with the solid line). The value of location state j is the same as the time t, so the j may represent the number of steps of migration to the VEGF source. Let the probability of cell lying at location state (i, j) at time t be P{ξ(t) = (i,j), t ∈ T} = p(i,j)(t), and p(i,j)(t) at first time (t = 0), when the cell has not migrated from the parent blood vessel, this is considered as 1. Moreover, we suppose that the angiogenic sprouts from the parent blood vessel are periodically distributed, s denotes the spacing between them, and N is a parameter related to the total number of the angiogenic sprouts. In this way, the probability p(i,0)(0) of initial location state, when the cells are located in each discrete position of the parent blood vessel, may be expressed as:
pði;0Þ ð0Þ ¼ p0 ¼

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The cell migration guided by the chemoattractant has certain randomness, and the fibronectin (FN) secreted by tip EC facilitates cellular adhering migration, but the concentration gradient of FN in the matrix of avascular tissue cannot be formed, so the haptotaxis effect of FN (Li et al., 2005) may be ignored. Thus we consider that the probability of cell moving to the front is the largest, and those to the oblique front both left and right are small and symmetrical. Let Q(i,j)(m,j + 1) represent probability of migration from (i,j) to (m, j + 1) (m ∈ [i − 1, i, i + 1]) in a unit time interval, Q(i,j)(i,j + 1) is maximum, Q(i,j)(i − 1,j + 1) and Q(i,j)(i + 1,j + 1) are small, and Q(i,j)(i − 1,j + 1) = Q(i,j)(i + 1,j + 1), Q(i,j)(i − 1,j + 1) + Q(i,j)(i,j + 1) + Q(i,j)(i + 1,j + 1) = 1. In this way, the probabilities of a cell migrating to the location ξ(i,j)(t) (Fig. 3, arrows with the dashed line) may be expressed as

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pði; jÞ ðt Þ ¼

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1 i ¼ ns; n ¼ 0; 1; 2; …N : 0 other

p0 t¼0 pði−1; j−1Þ ðt−1Þ Q ði−1; j−1Þði; jÞ þ pði; j−1Þ ðt−1Þ Q ði; j−1Þði; jÞ þ pðiþ1; j−1Þ ðt−1ÞQ ðiþ1; j−1Þði; jÞ

Where p(i,j)(t)(t ∈ T) satisfy the condition

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The Eqs. (2) and (3) show that the probability of location state reduces as the time t increases, implying that the distribution of migrating cells is increasingly dispersed along the direction i. In order to determine the distribution and main migration path of the cells, we perform an amplification processing for p(i,j)(t)(i ∈ (− ∞, …, − 1, 0, 1, …, ∞), j = t) to make the maximum of the p(i,j)(t) at time t become 1, and the transformed p(i,j)(t)is called equivalent probability in our calculation, so as to compare differences in probability between the location states at the same time. Suppose there are 5 points of sprouting in the parent blood vessel, and denote the spacing between them by s, s = 4. Because the higher concentration of chemokines leads to a reduced chemotactic sensitivity (Anderson and Chaplain, 1998), when the concentration of VEGF is high, the values of the migration probabilities Q(i,j)(i,j + 1), Q(i,j)(i − 1,j + 1), and Q(i,j)(i + 1,j + 1) approximate each other. To investigate the impact of the concentration of VEGF on migration characteristics of location state of ECs in the two groups. The migration probability of group 1 is

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pði; jÞ ðt Þ ¼ 2N þ 1; j ∈ ½0; 1; ⋯; ∞Þ:

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Fig. 3. The random directional migration of cells in the lattices. ξ(i,j)(t) represents the location of cell on lattice point (i, j) at time t.

Please cite this article as: Zhan, K., et al., Role of vascular endothelial progenitor cells in construction of new vascular loop, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.06.010

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and the migration probability of group 2 is

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The group 1 and group 2 represent the microenvironments with low and high intensity of VEGF, respectively, and the spacing s of the two groups are set to the same value for the present. Figs. 4 (a) and (b) show the equivalent probabilities of each location state for the two groups in the computational domain. The deeper staining means the large equivalent probability, and lighter staining means small equivalent probability. The horizontal axis (location state i) is parallel to the parent blood vessel, and the vertical axis (location state j) is parallel to the concentration gradient of VEGF. Fig. 4 indicates that there is a difference in equivalent probability at the same location state j, and the difference tends to decrease gradually as the j increases. In addition, the fusions of equivalent probabilities of location states in the two groups appear at the locations of j = 9 (t = 9) and j = 6 (t = 6), respectively, so there are t* = 9 in group 1 and t* = 6 in group 2, if t* denotes the fusion time. According to the analysis of the chemotactic response of EPCs, we consider that the cluster of EPCs may exude from the middle of the parent blood vessel between two vascular sprouts with the spacing of s, which is quivalent to the horizontal spacing between the neighboring light staining sites in Fig. 4, and migrate towards VEGF with high concentration (see Fig. 2 (b)), if plenty are mobilized. And when t = t*, the probability of the free EPCs arriving at each location parallel to the parent blood vessel is approximately equal, so the cells will be arranged in a cord. Due to aggregation effect (Armstrong and Parenti, 1972), the EPCs may adhere near the sprout tip and may be naturally embedded in the gap between the sprout tips to aid in anastomosis of the vascular sprouts, and complete the differentiation into ECs. If the mobilization of EPCs is sufficiently effective in this process, we may consider that when t = t*, the anastomosis of the angiogenic sprouts will reach the peak.

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Results

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Results of immunohistochemistry

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CD133 is a marker of EPCs. In the sections subjected to immunohistochemical staining with anti-CD133 antibody, the positive cells should show brown particles in cellular membranes and cytoplasms. The immunohistochemical staining image at 5 days after implantation showed that there were CD133 positive EPCs in the wound bed. They were dissociated, or aggregated, or ring-shaped (arrows in Fig. 5 (a)), and were with strongly or weakly positive expressions. Fig. 5 (a) also showed that there were mature blood vessels constructed by CD133 negative ECs (arrowheads). At 6 days after implantation, the CD133 positive EPCs were observed within the silk fibroin material (arrows in Fig. 5 (b)), they were dissociated single cells, or aggregated cell clusters, or with small rings similar to lumen, the diameters of those rings were less than the diameter of a normal capillary.

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Fig. 4. Distributions of equivalent probabilities of location states of migrating cells. Deep staining means a large equivalent probability and light staining means a small equivalent probability. Various values are indicated by the colorbar. (a) Group 1. (b) Group 2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

TEM observation

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The TEM image (Fig. 6 (a)) of the silk fibroin material at 7 days after implantation showed two endothelial-like cells attached to each other and one isolated cell (arrow), they had lumen-like vacuoles (“L” in Fig. 6 (a)). In the TEM image (Fig. 6 (b)) at 16 days after implantation, a tip endothelial cell (“E” in Fig. 6 (b)) extending filopodia (arrowheads) and a hematopoietic stem cell-like cell with a large nucleus (“S” in Fig. 6 (b)) were observed. A lamellipodia (arrow) of the hematopoietic stem cell-like cell was opposite to the direction of the filopodia, exhibiting a tendency of the two cells to get close to each other.

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Simulation of new vascular loop formation

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In the random directional migration process, the tip ECs on angio- 248 genic sprouts may either naturally join to form into a new vascular 249

Please cite this article as: Zhan, K., et al., Role of vascular endothelial progenitor cells in construction of new vascular loop, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.06.010

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loop or cannot join, keeping certain spacing. In addition, a cluster of EPCs either may fill the gap between two sprout tips to aid in anastomosis of the vascular sprouts or continues migrating to the chemokine source, crossing over a large gap. Thus, we consider that the behaviors of chemotactic migration of ECs/EPCs are implicated in two manners of new vascular loop formation: “natural anastomosis” and “EPC-assisted anastomosis” of the angiogenic sprouts. Set 20 points as the sprouting locations in the parent vessel. Let the spacing between the sprouts be s = 4 (dimensionless), and make a cluster of EPCs (e.g. 6 cells) migrate out from the middle of the parent vessel between two neighboring sprouts. Using the migration probability of group 1, we simulate the migration behaviors of the tip ECs and EPCs. In the simulation test, when two tip ECs encounter at time t b t*, we consider that the vascular sprouts are naturally joined, leading to a new vascular loop, defined as “natural anastomosis”; when the spacing between two neighboring tip ECs, L = 1 (t b t*), we consider that the EPCs are able to assist in joining the vascular sprouts, defined as “EPC-assisted anastomosis”; when t = t*, (s = 4), we consider that the EPCs arranged in cord are able to fill the gap between the two neighboring tip ECs to join them by timely proliferation, defining as “EPC-assisted anastomosis” in the fusion time, and if L N s (s = 4) at this time, we consider that the number of the mobilized or recruited EPCs is unable to fill the large gap, so a vascular loop cannot be formed. Fig. 7 shows two random results of simulating new vascular loop formation by computer program using the migration probability of the group 1.

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Fig. 5. Immunocytochemical images of CD133. (a) In the wound bed at 5 days after implantation, the CD133 positive EPCs (arrows) were dissociated, or aggregated, or ring-shaped, and the CD133 negative ECs constructed mature blood vessels (arrowheads). Bar: 50 μm. (b) In the porous silk fibroin film at 6 days after implantation, the CD133 positive EPCs (arrows) were dissociated single cells, or aggregated cell clusters, or with small rings similar to lumen. Asterisks and triangles indicate the sites of material and pores, respectively. Bar: 50 μm.

Fig. 6. TEM images of the porous silk fibroin film (the material is not shown). (a) 7 days after implantation. Arrows indicate endothelial-like cells with lumen-like vacuoles denoted by L. Bar: 1 μm. (b) 16 days after implantation. E: tip endothelial cell; S: hematopoietic stem cell-like cell; L: lumen; arrow: lamellipodia; arrowheads: filopodia. Bar: 2 μm.

In Fig. 7, 20 vascular sprouts (ploy lines) grow out from the parent blood vessel (thick line) and are discontinued at the sites of either natural anastomosis or EPC-assisted anastomosis, or continue to grow in the form of single sprout. The squares mark the sites of natural anastomosis, and a series of circles as EPCs (6 cells, overlapped) are shown at the sites of EPC-assisted anastomosis. The circles, whose ordinates are less than 9, join two neighboring sprouts with a spacing L = 1, and other circles, whose ordinates are equal to 9, join two neighboring sprouts with a spacing L ≤ 4.

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Rates of the natural and EPC-assisted anastomosis in new vascular loop 285 To further determine the role of EPCs in the construction of new vascular loops, we performed simulation tests 20,000 times (in this condition the variation of anastomosis frequencies with migrating step tended to be stable) and counted the number of the anastomosis frequencies at each time point. The counting is divided into two cases: (1) natural anastomosis by the contact of the neighboring tip ECs and (2) EPC-assisted anastomosis by the EPCs being embedded in the gap between two sprout tips. Based on the migration probabilities of Eqs. (4) and (5), we obtained the anastomosis frequencies of group 1 and group 2.

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(1) To use migration probability of the group 1 296 Fig. 4 (a) shows that the fusion time of equivalent probabilities 297 of location states is t * = 9. When there is no participation of 298

Please cite this article as: Zhan, K., et al., Role of vascular endothelial progenitor cells in construction of new vascular loop, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.06.010

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EPCs, the anastomosis of vascular sprouts is all natural, the distribution of the anastomosis frequencies is shown in Fig. 8 (a), whereas when there is participation of EPCs, the anastomosis of vascular sprouts is divided into two kinds of natural and EPC-assisted anastomosis, their frequencies are shown in Figs. 8 (b) and (c), respectively. Fig. 8 (a) shows that the frequency of natural anastomosis gradually increases as the number of migration steps increase, which is correlative to the distribution of tip ECs becoming more and more dispersed. The maximum of anastomosis frequencies appears in step 9 of the EC migration and is equal to 6639, meaning that one pair among the 20 sprouts (10 pairs) may be naturally joined in step 9 through about 3.01 simulation tests. The total anastomosis frequency until step 9 is equal to 36,225. If an anastomosis frequency that 20 sprouts (10 pairs) are all lined through the 20,000 tests is set as a baseline, the rate of the natural anastomosis until step 9 should be 36,225 / (20,000 × 10) = 18.11%. Figs. 8 (b) and (c) show distributions of the frequencies of the natural and EPC-assisted anastomosis, respectively, when there is participation of EPCs. The maximum of the frequencies of the natural anastomosis appears in step 5 and is equal to 1129. Investigating all natural anastomosis, the total anastomosis frequency until step 9 is equal to 7014, meaning that the rate of the natural anastomosis is equal to 3.51%. Fig. 8 (c) shows that the maximum of the frequencies of the EPC-assisted anastomosis is equal to 11,021, which appears in step 5. In this figure, another peak of the anastomosis frequency appears in the probability fusion time t* = 9. Thus, until t* and t* − 1, the total frequencies of EPC-assisted anastomosis are 143,411 and 62,849, respectively, the rates of anastomosis are 71.7% and 31.42%, respectively. (2) To use migration probability of the group 2 Fig. 4 (b) shows that the fusion time of location state equivalent probabilities is t * = 6. In the case of no participation of EPCs, the distribution of the frequencies of the natural anastomosis in 20,000 simulations is shown in Fig. 8 (d), and in the case of participation of EPCs, the distributions of frequencies

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299 300

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Fig. 7. Two random results of simulating new vascular loop formation. The 20 angiogenic sprouts extend from the horizontal parent vessel (thick line), and the tip ECs migrate toward the VEGF with high concentration to form the curved trajectories (poly lines). When the neighboring sprout tips contact, a vascular loop is formed by natural anastomosis and the anastomosis site is marked by a square; when the distance between the neighboring sprout tips affords the conditions of EPC-assisted anastomosis, a vascular loop is formed by EPC-assisted anastomosis, a cluster of EPCs, 6 cells, overlapped, denoted by the circles, shows at their migration site and the joined sprouts stop extending. Other vascular sprouts continue to extend in the form of single-vessel.

of the natural and EPC-assisted anastomosis are shown in 337 Figs. 8 (e) and (f), respectively. 338

In the two groups, the total sums of anastomosis frequencies in 20,000 simulation tests are counted, according to both the natural anastomosis in the case of no participation of EPCs and the natural and EPC-assisted anastomosis in the case of participation of EPCs. The rates of anastomosis under different conditions are shown in Table 1.

339

Discussion

345

340 341 342 343 344

According to the results of the animal experiments and numerical 346 simulations, we discuss the behaviors of EPCs and characteristics of 347 vasculogenesis in the adult from four aspects. 348 (1) EPCs participate in construction of new blood vessel The bone marrow-derived EPCs are immature vascular endothelial cells, whose properties of directional homing, differentiation in situ and participating in angiogenesis have been confirmed by a large number of previous studies (Isner and Asahara, 1999; Nolan et al., 2007; Werner et al., 2007; Gao et al., 2008; Asahara et al., 2011). In this study, taking biomaterial scaffold implanted into damage skin tissue of rat as an object of the study, we investigated the performance characteristics of EPCs in the regenerative tissue by immunohistochemical method. The dissociated, aggregated and ring-shaped CD133 positive EPCs (Fig. 5 (a)) showed that the EPCs participated in neovascularization of the wound bed and biomaterial, and the weakly positive expressions explained that the EPCs were preparing differentiation into ECs lining the new blood vessels, because the specific marker CD133 of EPCs is able to disappear after they differentiate into ECs (Peichev et al., 2000). (2) EPCs assist in anastomosis of angiogenic sprouts The microscopic images from the experiments can only describe some fragments of the biological behaviors, so it is very difficult to explore the process of EPCs participate in angiogenesis, whereas the numerical simulations for the cell

Please cite this article as: Zhan, K., et al., Role of vascular endothelial progenitor cells in construction of new vascular loop, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.06.010

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Fig. 8. Statistical data of the anastomosis frequency of 20 vascular sprouts in 20,000 simulation tests. (a) The case of no participation of EPCs (group 1). (b), (c) The case of participation of EPCs (group 1). (d) The case of no participation of EPCs (group 2). (e), (f) The case of participation of EPCs (group 2).

Please cite this article as: Zhan, K., et al., Role of vascular endothelial progenitor cells in construction of new vascular loop, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.06.010

K. Zhan et al. / Microvascular Research xxx (2013) xxx–xxx

384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426

71.70 31.42 69.73 27.56

75.21 34.54 75.14 31.99

= 9) − 1 = 8) = 6) − 1 = 5)

movements can reflect the process of cell behaviors. In this study, we simulated the chemotactic migrations of the tip ECs and EPCs, based on the hypothesis that the concentration gradient of VEGF in avascular tissue may induce sprouting of the vascular plexus, which wraps around the tissue, and the EPCs can migrate into the avascular tissue from the peripheral blood. The results show that the angiogenic sprouts may connect by way of two sprout tips joining to be a blood supply loop, and that a cluster of EPCs may fill the gap between two sprout tips to become a vascular anastomosis segment by differentiation and proliferation, when plenty of EPCs are mobilized, see Fig. 7. The EPCs in peripheral blood may be derived from different precursor cells such as multipotent adult progenitor cell (MAPC) (Reyes et al., 2002), and may be derived from hematopoietic stem cells (HSCs), because they have the same precursor called as hemangioblast and specific marker of CD133 (Isner and Asahara, 1999). Therefore, the hematopoietic stem cell-like cell denoted by S, which was close to the filopodia (Fig. 6 (b)), might be a precursor of vascular endothelial cell, and would be an elongation of the angiogenic sprout. The shapes of the cells in Fig. 6 (a) were closely analogous to the area denoted by a rectangle in Fig. 5 (a), which showed that the EPCs had lumen-like vacuoles, whose diameters were less than the normal diameter of capillary. Kamei et al. (2006) have indicated that intracellular and intercellular fusion of endothelial vacuoles drives vascular lumen formation. Combining the results of the numerical simulations, we may infer that the vacuolization of EPCs adjacent to the sprout tips means that the EPCs are entering a stage of lumen formation. (3) Mobilization of EPCs promotes vascularization in avascular tissue. Neovascularization of avascular tissue is gradually penetrated from the host (Zhan et al., 2010). For new vascular loops at the leading edge, the rates of natural and EPC-assisted anastomosis in Table 1 demonstrates that there is no significant difference in anastomosis rate between the groups within their respective fusion time t*. For instance, in the case of no participation of EPCs, the rates of natural anastomosis of two groups are 18.11% and 16.75%, respectively, and in the case of participation of EPCs, the total rates of anastomosis of two groups are 75.21% and 75.14%, respectively. This implies that similar percentage of the angiogenic sprouts may be joined to form a new blood supply loop in a natural time cycle depending on the microenvironment, and other single blood vessels will continue extending, degenerating or branching in response to ischemia and hypoxia. However, because the formed vascular loops will continue constructing next vascular loops, the group 2 with a small t* will form more new vascular loops within a same time. This also reflects an objective fact of the high concentration of chemokine leading to the high vascular density and vascularization degree. The rates of anastomosis in the Table 1 show that there is significant difference in the total rate of anastomosis between the two cases of participation and no participation of EPCs.

(until t* (until t* (until t* (until t*

= 9) − 1 = 8) = 6) − 1 = 5)

(until t* (until t* (until t* (until t*

= 9) − 1 = 8) = 6) − 1 = 5)

For instance, the total rates of anastomosis in group 1 are 18.11% and 75.21%, respectively, and those of group 2 are 16.75% and 75.14%, respectively, about 5-fold increase. Even if the peak of anastomosis in the fusion time t* is overlooked, the total rates of anastomosis of the two groups in the case of participation of EPCs amounted to 34.54% and 31.99%, respectively, they are approximately twice as large as those in the case of no participation of EPCs. This implies that the EPCs play an important role to assist in anastomosis of vascular sprouts during the construction of new blood supply loops. In the case of participation of EPCs, supposing that the length of one vessel segment is proportional to the number of cells in the vessel, the average ratio K of the number of the EPCs to that of ECs in one vascular loop may be expressed as

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(until t* (until t* (until t* (until t*

t X

K¼

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382 383

3.51 3.12 5.41 4.43

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380 381

Total rate of anastomosis (%)

E

378 379

16.75 (until t* = 6)

Rate of EPC-assisted anastomosis (%)

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376 377

2 (t* = 6)

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374 375

18.11 (until t* = 9)

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372 373

1 (t* = 9)

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t1:5 t1:6 t1:7 t1:8

Rate of natural anastomosis (%)

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Rates of anastomosis (the case of participation of EPCs)

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Rate of natural anastomosis (the case of no participation of EPCs) (%)

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t1:3

Table 1 The rates of natural and EPC-assisted anastomosis in 20,000 simulation tests.

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t1:1 t1:2

9

i¼1 t X

427 428 429 430 431 432 433 434 435 436 437 438 439 440

LAi :

ð6Þ

LSi

i¼1

Where LAi represents the total length of the anastomosis segments formed by the EPCs in step i by 20,000 simulations, t* is the fusion time of equivalent probability of location state. In step t*, the average length of the anastomosis segments is counted as 3 and the others are 1. LSi represents the total length of the angiogenic sprouts in all vascular loops formed in step i by 20,000 simulations. According to the simulation data (Fig. 8), we calculate K of group 1 and group 2 at 13.96% and 20.72%, respectively. If the peak of anastomosis in the fusion time t* is overlooked, the K of the two groups are 8.60% and 11.60%, respectively. This implies that a very small amount of EPCs can play a role to accelerate the formation of a new blood supply loop, and that the higher concentration of VEGF in avascular tissue, such as group 2, can cause an increased contribution of EPCs, which is consistent with the findings from other sources: EPCs derived from circulating hematopoietic progenitors were 8.3– 11.2% of ECs in vessels of granulation tissue (Crosby et al., 2000), and the degree of contribution of BM-derived EPCs to tumor angiogenesis was 35–45% (Reyes et al., 2002). Therefore, when the tissue microenvironment mobilizes plenty of EPCs homing, the efficiency of vascularization will be greatly improved, on the other hand, the excessive endogenous EPCs will lead to vascular hyperplasia, contributing to the abnormal growth of the tissue. (4) Similarity and difference between the adult and embryo in manner of vasculogenesis The biomaterial scaffold implanted into the adult tissue is a novel experimental model used to study vasculogenesis in the adult, due to the neovascularization process from nothing to something. Vasculogenesis during embryogenesis is that undifferentiated precursor cells aggregate into a blood island and differentiate in situ to mature ECs giving rise to a primary capillary plexus

Please cite this article as: Zhan, K., et al., Role of vascular endothelial progenitor cells in construction of new vascular loop, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.06.010

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485 486 487 488 489

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(Risau et al., 1988; Carmeliet, 2000). In this study, based on the mutual corroboration of the results of experiments and numerical simulations, we propose that the EPCs play a role in assisting in anastomosis of vascular sprouts in construction of new vascular loop. The process is: EPCs exude from blood vessels, migrating to the site of angiogenic sprout tips, aggregating, becoming a cell cord, and differentiate in situ to ECs during the cell vacuolization and vacuole fusion, finally an anastomosis segment of the vascular sprouts is formed (see Fig. 9). Therefore, the similarity between the adult and embryo is that vasculogenesis is dependent on EPCs aggregating and differentiating in situ, but the difference is that vasculogenesis in the adult occurs mainly in the anastomotic site of angiogenic sprouts.

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Fig. 9. Anastomosis of the angiogenic sprouts. (a) Natural anastomosis. The contact of the neighboring sprout tips leads to anastomosis of the sprouts. (b) EPC-assisted anastomosis. The EPCs exuding from the existing blood vessel migrate to the site of sprout tips, aggregating, differentiating, finally become an anastomotic vessel segment of the angiogenic sprouts.

Conclusion

491

Sprouting angiogenesis is a main mode of vascularization of avascular tissue. In this study, we find that the EPCs can play a role to assist in anastomosis of angiogenic sprouts, when plenty of EPCs are mobilized from bone marrow into the avascular tissue. By the experiments and numerical simulations complementing and confirming each other, we conclude that (1) the EPCs participate in the construction of new capillaries, because the CD133 positive EPCs with lumen-like vacuoles were detected in the regenerative skin tissue of rat, (2) a small number of EPCs is able to lead the newly formed blood vessels to fast functionalization, because the rates of the EPC-assisted anastomosis far exceeded the rates of the natural anastomosis and the average ratio of the number of the EPCs to ECs in one vascular loop was small, and (3) the manner of vasculogenesis in the adult tissue is similar to that during embryogenesis, such as EPCs aggregating and differentiating in situ, but this behavior of EPCs in the adult tissue occurs in the anastomotic site of angiogenic sprouts.

494 495 496 497 498 499 500 501 502 503 504 505 506

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492 493

N

490

Acknowledgment

507

We thank Mingzhong Li for assisting with the material preparation; Baoqi Zuo, Liang Xie and Xinhong Wang for assisting with the animal experiments. We also acknowledge the reviewers for their exhaustive and useful comments. This work was partially supported by the National Key Basic Research and Developing Project of China: Basic Research of the Tissue Inducing Biomaterial Used in Medicine (Project no. 2005CB623906).

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