In general, normal tissues have a barrier, preventing either endothelial cell migration or tumour cell invasion. The effect of this barrier can be interrupted by newly-formed stroma, namely stromatogenesis, during the process of tumour development . Stromatogenesis is probably a response to messages delivered by tumour cells. The newly-formed stroma is usually loose and oedematous and therefore allows endothelial and tumour cells to easily penetrate it .
Angiogenesis in relation to clinicopathological variables
It has been found that high microvessel density (MVD) is associated with VEGF [25–27] and VEGF-C expression at the deepest invasive tumour site [24, 28]. However, high MVD is not associated with VEGF-D expression . Several studies have demonstrated that MVD gradually increases from normal mucosa to adenoma and finally to carcinoma in the colorectum [29–33]. Increased MVD is detected at the early stages of focal dysplasia, and then increases gradually from low to high grades of dysplasia . The transitional mucosa adjacent to the carcinoma displays intermediate levels of MVD between normal mucosa and the carcinoma . In the carcinoma, MVD increases as the tumour invades from the mucosa to the muscularis propria . The highest level of MVD is found at the invasive margin of carcinomas , a site of active tumour invasion. These findings indicate that MVD is an early and critical step in colorectal tumourigenesis and tumour development.
Many research groups have studied CRC and shown that a high grade of MVD is related to a larger tumour size [12, 35, 36], non-mucinous carcinoma , poorer differentiation [12, 34], deeper invasion of tumours , advanced Dukes' stage [12, 34], lymphatic vessel invasion [34–36], lymph node metastasis [31, 34, 36], venous vessel invasion [34–37], liver metastasis , and a higher rate of recurrence [36, 38–40]. Tumours with high levels of MVD have been connected to poor survival in patients with earlier or advanced colon/rectal cancers [12, 27, 40–42]. Even in multivariate analyses, MVD is related to survival in the whole group of patients with CRC [34, 36, 38, 43] or subgroups of patients with stage II-III  or stage A-C tumours . Recently, Yonenaga et al. have analysed a microvessel pericyte coverage index (an index of microvessel maturation) in relation to clinicopathological significance in CRC . The results indicate that immature neovascularization is observed in poorly differentiated tumours and further correlated with metastasis, resulting in a poorer prognosis. Thus, not only microvessel density but also vessel maturation are crucial factors for the tumour development and aggressiveness of CRC. Notably, Yonenaga et al. have applied the anti-α-smooth muscle actin (SMA) marker to determine the microvessel pericyte coverage index . There are several other common markers used for pericytes including desmin, PDGFR-β, VEGFR-1, and neuron-glial antigen 2. It seems that α-SMA and desmin expression are esentially identical in pericytes [12, 45]. While PDGFR-β, VEGFR-1, and neuron-glial antigen 2 can be positively expressed in pericytes that are negative for α-SMA, indicating that a lack of α-SMA expression does not necessarily mean an absence of pericytes. In other words, PDGFR-β, VEGFR-1, and neuron-glial antigen 2 may be more reliable markers for determining the presence of pericytes, while α-SMA is probably only expressed in more stable and mature pericytes .
There are a few conflicting reports in CRC regarding the clinicopathological significance of MVD, in which MVD is not associated with tumour stage [29, 43, 47], vascular and neural invasion , metastasis , or survival in the whole group of patients [4, 29, 48, 49], subgroups of patients in stage A-C , or patients with stage I and II rectal cancer . In addition, there were four studies which are performed on a small number of colon and/or rectal cancers (from 22 to 48 cases) that also show a non-association of MVD with the clinicopathological variables including tumour size, location, grade of differentiation, the presence of a mucinous component, stage, vascular or lymphovascular or neural invasion, or patient survival [30, 51–53].
There are even opposite results from two studies in which higher values of MVD appear to be in the early stages of CRC , and correlate with longer disease-free survival and overall survival in patients with node-negative CRC . Recently, Peeters et al. observed an increased vascularization of metastases in the liver after resection of the primary CRC . This result suggests that the primary tumour may produce certain circulating inhibitors of angiogenesis that suppress the angiogenesis of metastases. Therefore, after resection of the primary tumour, the circulating levels of this inhibitor decrease, resulting in increased angiogenesis and, as a consequence, growth of the metastases .
Hypercoagulation in cancer patients is another factor for tumour progression. Substantial evidence from preclinical experiments and clinical practice has supported the association between activation of blood coagulation and progression of the cancer. Cancer patients display a wide range of coagulation disorders from asymptomatic laboratory changes to massive thromboembolism and disseminated intravascular coagulation. About 50% of all cancer patients and 90% of patients with metastasis have abnormalities in coagulation tests. CRC is the second most common cancer diagnosed in patients with thromboembolic events. Blood vessel thrombosis leads to impairment of blood flow, ischemia, and organ damage. The hemostatic complications are the second most common cause of death in cancer patients .
Studies have shown that plasma D-dimer levels, representing activation of coagulation and fibrinolysis, are increased in most patients with CRC compared to patients with benign colorectal disease. Furthermore, the D-dimer level is positively related to tumour size, wall penetration, lymph node invasion, and hepatic metastasis . In a multivariate analysis, the D-dimer level in preoperative plasma in CRCs is the third strongest prognostic factor, after lymph node status and preoperative carcinoembryonic antigen level . Fibrinolytic capacity was much higher in advanced CRCs, indicating a progression to overt disseminated intravascular coagulation .
Although the mechanism behind hypercoagulation in cancer patients is unclear, the main factor responsible for hypercoagulation has been considered to be cancer itself. It has been shown that tumour cells activate the coagulation system by producing and secreting procoagulant/fibrinolytic substances and inflammatory cytokines, as well as physically interacting with blood (monocytes, platelets, neutrophils) or vascular cells . This activation is accompanied by the consumption and decline of coagulation inhibitors. Other mechanisms for hypercoagulation in cancer patients include non-specific factors such as the generation of acute phase reactants, necrosis, abnormal protein metabolism and hemodynamic compromise. In addition, anticancer therapy may also increase the risk of blood coagulation by similar mechanisms, e.g., release of procoagulant/fibrinolytic substances and inflammatory cytokines, damage of endothelial cells, and stimulation of tissue factor production by host cells .
A recent study shows that VEGF is highly expressed in primary CRC compared to the corresponding adjacent normal mucosa . VEGF expression appears to be absent in mild to moderate dysplasia adenomas of the colorectum, and is present in the majority of carcinomas-in-situ and in all carcinomas invading the submucosa . VEGF-D is more highly expressed in carcinoma than in the adjacent normal mucosa [22, 24] and adenoma , while VEGF-C expression in normal mucosa does not differ from that in CRC . Notably, one study shows that VEGF-D expression is significantly lower in both polyp and carcinoma compared to normal mucosa while VEGF-A and VEGF-C are significantly raised in carcinoma compared to normal mucosa and polyp. One explanation for this is that decreased VEGF-D may allow for higher levels of VEGF-A and VEGF-C to bind more readily to the VEGF receptors, producing the angiogenic switch required for tumour growth .
Increased expression of VEGF-A in CRC is associated with lymphatic metastases . Increased VEGF-C expression correlates significantly with poorer differentiation , deeper invasion of tumours [28, 63], advanced Duke's stage , lymphatic invasion, lymph node metastasis [28, 63], venous invasion , and liver metastasis [28, 64]. VEGF-D is associated with lymphatic involvement . Overall, high VEGF expression is related to larger tumour size [65, 66], non-mucinous carcinoma , advanced stage [65–67], blood vessel invasion, liver metastasis , multiple numbers of metastases , and recurrence .
There are few studies of VEGFR expression in CRC. Some studies show that either VEGFR-2 or -3 expression on CRC does not differ from that in the normal mucous of the colorectum [19, 22], while others show that VEGFR-3-positive vessel densities increase progressively from normal mucosa to adenoma and to carcinoma [22, 24, 69]. Furthermore, VEGFR-3 is associated with lymph node metastasis .
There are controversial results regarding the role of VEGF and VEGFR in CRC. For example, levels of VEGF expression in primary CRC and liver metastases do not significantly differ . VEGF-A has no impact on patient survival . VEGF-C is not related to gender, histological type, venous involvement , lymph node invasion , liver metastasis or survival . VEGF-D and VEGFR-3 expression do not correlate with grade of differentiation, Dukes' stage (A to C) or survival .
Even splicing variants in certain members of the VEGF family play different roles in tumour development. For example, the VEGF-A gene, located on chromosome 6p21.3 with eight exons, gives rise to several distinct isoforms of VEGF-A through alternative mRNA splicing. The more common isoforms of human VEGF-A consist of VEGF121, VEGF145, VEGF165, VEGF165b, VEGF189, and VEGF206, and other isoforms such as VEGF148, VEGF162 and VEGF183 have also been reported. VEGF-B also has different isoforms such as VEGF167 and VEGF186 [70, 71]. These isoforms differ in their expression patterns as well as their biochemical and biological properties. In normal colonic tissue, VEGF121 and VEGF165 are mainly expressed, whereas VEGF189 is expressed rarely and weakly. VEGF121 and VEGF165 are diffusible secreted proteins with low affinity to heparin, whereas VEGF189 and VEGF206 have a high affinity to heparin-like molecules such as heparansulfate . Okamoto et al. examined the expression patterns of several VEGF-A isoforms in 228 established xenografts originating from various human solid tumours including colon cancer. VEGF121/VEGF165 were seen in 27 xenografts and VEGF121/VEGF165/VEGF189 in 201 xenografts. VEGF189 is more frequently expressed in all tumour xenografts than in primary tumours, indicating that VEGF189 contributes to the successful xenotransplantability of various solid tumours through the induction of stromal vascularization . Although the rate of tumour growth depends on the level of VEGF expression, certain isoforms play a greater role in angiogenesis than others. VEGF165b inhibits VEGF165-mediated proliferation, migration of endothelial cells, and vasodilatation of mesenteric arteries. VEGF165b-expressing tumours grow significantly more slowly than VEGF165-expressing tumours. Thus, VEGF165b is an effector of anti-angiogenesis and is downregulated in certain tumours. These results suggest that regulation of VEGF splicing is a critical switch from an antiangiogenic to proangiogenic phenotype [74, 75].