During embryonic development, angioblasts migrate to various regions of the developing embryo and differentiate into ECs in response to local cues such as growth factors and ECM components. The ECs then form a vascular plexus, which is a network built by connections (anastomoses) between blood vessels. This process is called vasculogenesis and it is not limited to the embryonic period, as a similar process can also occur in adults through the recruitment and participation of bone marrow-derived endothelial progenitor cells [3, 7, 8].

Angiogenesis is another mechanism of blood vessel formation that occurs through the sprouting of existing blood vessels. Angiogenesis is a sequential, multistep process that begins with activation of a quiescent endothelium by proangiogenic factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and angiopoietin-2 (Ang2) that are often produced by hypoxic or tumorigenic tissues [3, 8]. Hypoxia up-regulates expression of a number of genes involved in vessel formation, patterning, and maturation, including nitric oxide synthase, VEGF, and Ang2. Nitric oxide, which is a product of nitric oxide synthase, dilates vessels and make them more responsive to VEGF because they becomes more leaky. Ang2 also facilitates sprout formation in the presence of VEGF. The sprouts anastomose to form vascular loops and networks [3].

Angiogenesis is also dependent on degradation of the BM, a thin layer of ECM between the epithelial cell layer and the endothelial cell lining of blood vessels. Degradation of BM is due to up-regulation of matrix metalloproteinases (MMPs) such as MMP2, MMP3, and MMP9, and suppression of protease inhibitors such as tissue inhibitor of metalloproteinase-2 (TIMP2). BM degradation is followed by migration of an endothelial tip cell from the leading edge of a vascular sprout; this leading edge defines the direction of the newly growing sprout [2, 6, 7].

High levels of proangiogenic factors (such as VEGFA and VEGFC) and of VEGF receptor 2 (VEGFR2) or VEGFR3 signaling select “tip cells” (TCs) for sprouting during angiogenesis. By contrast, Delta-like 4-notch signaling laterally inhibits TC fate in adjacent ECs. TC sprouting behavior is facilitated by the vascular endothelial cadherin-mediated loosening of EC–EC junctions, matrix metalloproteinase-mediated degradation of ECM and the detachment of pericytes. Guidance of TC sprouting is due to the gradients of proangiogenic growth factors and various environmental guidance cues, such as semaphorins and ephrins. During sprout elongation, TCs are trailed by endothelial “stalk cells” (SCs), which maintain connectivity with parental vessels and initiate partitioning-defective3 (PAR3)-mediated vascular lumen morphogenesis. Expression of VEGFR1 and activation of notch, Roundabout homologue 4 and WNT signaling in SCs repress TC behavior to maintain the hierarchical organization of sprouting ECs. However, TCs and SCs may also shuffle and exchange positions during angiogenic sprouting. Upon contact with other vessels, TC behavior is repressed and vessels fuse by the process of anastomosis, which is assisted by associated myeloid cells. The ECs adjacent to the tip cells begin to proliferate and elongate to form capillary sprouts, which then assemble to form a vessel lumen. After the activation and proliferation stages, a nascent blood vessel must mature to become functional [6–11].

After the activation and proliferation stages, a nascent blood vessel must mature to become functional. The nascent vessels are stabilized by recruiting mural cells and by deposition of an ECM, in a process known as arteriogenesis. There are at least four molecular pathways that regulate this process, including the following:

Recruitment of mural cells such as pericytes and smooth muscle cells (SMCs) to the developing immature vasculature by platelet-derived growth factor B (PDGFB) and transforming growth factor-β1 (TGF-β1) stabilize the vessel wall [2, 7, 12, 13]. The contact between ECs and mural cells is strengthened by bioactive lipid spingosine1 phosphate (S1P) through activating the guanine nucleotide-binding-coupled receptor, and S1P receptor 1(S1PR1 or EDG1) signaling [7, 14]. Tie receptors (Tie1 and Tie2) and their ligands Ang1 and Ang2 are also critical for vessel formation and stabilization. Main sources of Ang1 and Ang2 are the mural cells and ECs, respectively. Ang1 stabilizes nascent vessels by facilitating communication between ECs and mural cells. Ang2 acts as an antagonist of Ang1 in the absence of VEGF and destabilizes vessels in the presence of VEGF [15].

TGF-β1 is a multifunctional growth factor that promotes vessel maturation by stimulating ECM production and by inducing differentiation of mesenchymal cells to mural cells. It is expressed in a number of cell types, including ECs and mural cells, and depending on the context and concentration, could be pro- or anti-angiogenic. Recent in vitro studies indicate that the TGF-β1–ALK1 pathway is a positive regulator of endothelial cell migration and proliferation by inducing ECs and fibroblasts to express Id1, a protein required for proliferation and migration. On the other hand, the TGF-β1–ALK5 pathway is a positive regulator of vessel maturation by inducing the plasminogen activator inhibitor (PAI1) in ECs. PAI1 promotes vessel maturation by preventing degradation of the provisional matrix around the nascent vessel. Thus, the degree to which TGF-β signals through ALK1 versus ALK5 can determine the pro- or anti-angiogenic effect of TGF-β [2, 10]. Figure 6.2 shows important signaling molecules that are involved in vasculogenesis, maturation of blood vessels and angiogenesis.

Human umbilical vein endothelial cells (HUVECs) show relatively higher proliferative potential among the isolated CD31⁺ ECs that originate from veins and arteries of different tissues [28]. Isolation of CD34⁺ and Flk-1⁺ cells from peripheral blood using magnetic beads is another source of adult ECs [16]. These isolated progenitor cells could differentiate into ECs and incorporate into neovascularization sites in mouse and rabbit hindlimb ischemic models [34]. CD34⁺ cells, mobilized from the bone marrow following treatment with granulocyte macrophage colony stimulating factor, improved ventricular function and neoangiogenesis in ischemic nude rat myocardium [35]. CD133⁺ cells purified from bone marrow were also shown to enhance human myocardial perfusion and global function [36]. Another important source of adult ECs is the umbilical cord blood, which contains more CD133⁺ and CD34⁺ cells than adult peripheral blood and has higher proliferation capacity [37].

Yamanaka et al. have shown systematic differentiation of cardiovascular cells from mouse iPSCs. Induced pluripotent stem (iPS) cells were generated from mouse skin fibroblasts by introducing four transcription factors (Oct3/4, Sox2, Klf4, c-myc), and then, the same approach was applied on ES cells to induce cardiovascular differentiation. They showed that Flk1⁺ cells could differentiate into artery, vein, and mural cells [38]. Rufaihah et al. have differentiated human iPSCs (hiPSCs) into endothelial cells (hiPSC-ECs) to assess their ability to improve perfusion in a murine model of peripheral arterial disease [39, 40]. In brief, endothelial differentiation was initiated by culturing hiPSCs for 14 days in differentiation media supplemented with bone morphogenetic protein 4 and VEGF. They purified the heterogenous mixture of cells by FACS using an antibody directed against CD31. The purified hiPSC-ECs generated capillary-like structures when grown in Matrigel and incorporated acetylated-LDL cholesterol. These cells expressed endothelial markers such as FLK-1 (KDR), CD31, CD144, and eNOS. When exposed to hypoxia, the hiPSC–ECs produced angiogenic cytokines and growth factors. Subsequently, they transduced the cells with a double-fusion construct comprising firefly luciferase for BLI and green fluorescence protein for histochemistry. The hiPSC–ECs were administered on days 0 and 7 after femoral artery ligation into the ischemic hindlimb of immunodeficient mice. Over a two-week period, BLI revealed reduction of cells, but some hiPSC–ECs survived in the ischemic limb for at least 2 weeks. At that time, and for up to 4 weeks, perfusion was improved by over 30 % by comparison to the vehicle-treated group, as assessed by laser Doppler imaging. This effect was associated with a 60 % increase in the total number of capillaries in the ischemic limb of mice receiving hiPSC–EC injections by comparison to the vehicle-treated group. This preclinical work provided proof-of-concept for the use of hiPSC–EC in peripheral arterial disease [41].

Figure 6.3 shows development of therapeutic cells from human-induced pluripotent cells. Readily accessible somatic cells (e.g., skin fibroblasts) are harvested from a patient and expanded in culture. Cells are exposed to reprogramming transcriptional factors, in the form of cell permeant peptides and modified mRNA. The resulting iPSC colonies are differentiated into vascular progenitor cells, that are administered directly to patients with vascular disease, or which are incorporated into matrices as a biological conduit such as cylindrical bioengineered matrix or decellularized cadaveric vessels for surgical implantation. These bioengineered conduits would serve to replace autologous saphenous vein in patients that have insufficient or diseased veins [41].

The approaches to engineer macrovessels can be divided into several distinct categories, although there may be some overlapping features. Successful application of these approaches, either individually or in combination, is expected to enhance therapeutic opportunities by building functional tissue and organ systems for regenerative medicine.

In all of the mentioned approaches, choosing the right cell among the spectrum of stem cells to differentiated cells and also the right scaffold is of utmost importance [42]. The scaffold should provide the initial requisite mechanical strength to withstand in vivo hemodynamic forces until vascular SMCs and fibroblasts reinforce the ECM of the vessel wall. Hence, the choice of scaffold is crucial for providing guidance cues to the cells to behave in the required manner to produce tissues and organs of the desired shape and size. Several types of scaffolds have been used for the reconstruction of blood vessels. They can be broadly classified as biological scaffolds, decellularized matrices, and polymeric biodegradable scaffolds. A review written by Pankajakhshan et al. focuses on the different types of scaffolds that have been designed, developed, and tested for tissue engineering of blood vessels [43].