**2. The angiogenic cascade**

Angiogenesis is the complex physiological sequence of vasodilatation, degradation of base‐ ment membrane, endothelial cell migration, chemotaxis, increasing vascular permeability and eventually endothelial cell proliferation and vessel formation. The fine-tuned balance of vasculo- and angiogenesis is controlled by many growth and transcription factors (Pandya, Dhalla et al., 2006).

#### **2.1. Angiogenesis versus vasculogenesis**

Lack of oxygen and nutrients threatens the tissue integrity and viability fundamentally. In these situations of undersupply, the organisms´ reaction is to improve the local perfusion by inducing the growth of the vasculature.

Angiogenesis is one crucial mechanism; it describes the sprouting of a vascular system on the base of pre-existing capillaries via endothelial migration and proliferation. This kind of vascular regeneration is the more common one.

When endothelial progenitor cells are mobilized from the bone marrow to differentiate and proliferate to form new vessel architecture, vasculogenesis takes place. An alternative way to vasculogenesis bears on the potential of local endothelial to differentiate and proliferate without a pre-existing vessel structure (Tepper, Cappla et al., 2005, Hankenson et al., 2005).

Both processes have in common that the smooth frictionless procedure depends on a continu‐ ing dynamic crosstalk between endothelial cells and surrounding connective tissue. Other important cell types are monocytes, macrophages, fibroblasts, pericytes or smooth muscle cells (Nyberg, Salo et al., 2008).

The biochemical and morphological roll out of vessel formation is standardized: angiogenesis starts with the vasodilatation of the original vessels followed by the degradation of the basement membrane, migration and proliferation of endothelial cells, their arrangement in luminal structures, loop formation and establishment of new basement membranes (Moulton, Folkman et al., 1998).

#### **2.2. VEGF pathway**

Thehealthyvasculatureisoneprerequisiteofeveryregenerativeprocess.Ititsgovernedbymany interacting signalling pathways, the VEGF pathway is considered as one of the most crucial ones; it is certainly the most investigated and understood one (Dyer, Portbury et al., 2010).

The first observed function of VEGF was its ability to enhance the permeability of tumour vasculature. Later its power as endothelial mitogen was described: VEGF attracts endothelial cells and promotes their differentiation, proliferation and survival. Today the role of VEGF as one of the angiogenic factors to keep up and promote vascular homoeostasis in the organism has become clear.

The striking significance of VEGF becomes obvious considering the fact that the first definitive markerproteinonripeningendothelialcellsintheyolksacistheVEGFreceptor2(VEGFR2orFlk 1). Under the influence of VEGF A these endothelial progenitors marked by VEGFR 2 form areas ofbloodislands;theseformationsarecharacterizedbyclustersofinitialerythroblastslinedbythe endothelial precursors (Park, Afrikanova et al., 2004, Pearson, Sroczynska et al., 2008).

VEGF-A exists in several splice variants, with different characteristics; VEGF120 for example is thought to be an especially diffusible isoform due to the lack of a heparin-binding domain.

Three relevant receptors transmit the signal of specific VEGF binding: VEGFR-1 (flt-1), VEGFR-2 (KDR/flk-1) and VEGFR-3 (flt-4). The ligand-receptor interaction leads to cellular response on the base of receptor phosphorylation (Autiero, Waltenberger et al., 2003).

One regulative factor is the appearance of a soluble VEGF receptor, VEGFR-1 (sFlt-1) that acts as a so-called VEGF trap, catching VEGF-A and so inhibiting the initiation of angiogenesis (Maynard, Min et al., 2003).

The VEGF pathway is summarized in the KEGG signalling pathway: in this survey it becomes obvious that the most important receptor on endothelial cells is VEGFR-2 to transmit the angiogenic information. Starting from there several cascades are initiated. Their common outcome is the up regulation of genes that accomplishes endothelial cell proliferation and migration, focal adhesion and cell survival.

Relevant pathways are the calcium-signalling pathway, the MAPK signalling pathway and the arachidonic acid metabolism.

#### **2.3. Role of endothelial cells**

Finally the role of pluripotent cells in modern tissue engineering concepts is summarized.

for this survey.

Dhalla et al., 2006).

**2. The angiogenic cascade**

454 Regenerative Medicine and Tissue Engineering

**2.1. Angiogenesis versus vasculogenesis**

inducing the growth of the vasculature.

(Nyberg, Salo et al., 2008).

Folkman et al., 1998).

**2.2. VEGF pathway**

vascular regeneration is the more common one.

Fundamental research with special respect to cell culture, immunohistochemistry, in vitro and in vivo trials, circulation modelling and gene expression profiling provides the scientific basis

Angiogenesis is the complex physiological sequence of vasodilatation, degradation of base‐ ment membrane, endothelial cell migration, chemotaxis, increasing vascular permeability and eventually endothelial cell proliferation and vessel formation. The fine-tuned balance of vasculo- and angiogenesis is controlled by many growth and transcription factors (Pandya,

Lack of oxygen and nutrients threatens the tissue integrity and viability fundamentally. In these situations of undersupply, the organisms´ reaction is to improve the local perfusion by

Angiogenesis is one crucial mechanism; it describes the sprouting of a vascular system on the base of pre-existing capillaries via endothelial migration and proliferation. This kind of

When endothelial progenitor cells are mobilized from the bone marrow to differentiate and proliferate to form new vessel architecture, vasculogenesis takes place. An alternative way to vasculogenesis bears on the potential of local endothelial to differentiate and proliferate without a pre-existing vessel structure (Tepper, Cappla et al., 2005, Hankenson et al., 2005).

Both processes have in common that the smooth frictionless procedure depends on a continu‐ ing dynamic crosstalk between endothelial cells and surrounding connective tissue. Other important cell types are monocytes, macrophages, fibroblasts, pericytes or smooth muscle cells

The biochemical and morphological roll out of vessel formation is standardized: angiogenesis starts with the vasodilatation of the original vessels followed by the degradation of the basement membrane, migration and proliferation of endothelial cells, their arrangement in luminal structures, loop formation and establishment of new basement membranes (Moulton,

Thehealthyvasculatureisoneprerequisiteofeveryregenerativeprocess.Ititsgovernedbymany interacting signalling pathways, the VEGF pathway is considered as one of the most crucial ones;

it is certainly the most investigated and understood one (Dyer, Portbury et al., 2010).

Endothelial cells are the cellular key element of angiogenesis and play a significant role in all crucial steps:

Endothelial cells are able to produce and release growth factors.

Endothelial cells express different growth factor receptors on their surface and are regulated by their impact.

Endothelial cells are actively involved in the dissolution of the surrounding matrix.

Simultaneously the migration adhesion and proliferation of endothelial cells continues.

Endothelial cells start to express characteristic integrins to anchor and pull forward the sprouting vessels.

Specialendothelialcells,thetipcellssecretematrixmetalloproteinasestopavethewayandloosen the connective tissue in front of the sprouting vessels´ tip to facilitate the further out growth.

**Figure 1.** KEGG VEGF signalling pathway (Homo sapiens)

Proliferating endothelial cells are capable of forming three-dimensional structures as tubes and loops, the structural fundament of a functioning circulation.

Considering all these key functions it becomes clearly obvious that only an intact endothelial property can effectively lead to angiogenesis and provides the prerequisite for any regenera‐ tive process (Pandya, Dhalla et al., 2006).

These special demands during vascular regeneration are reflected in a significantly increased turn over time. Normally the endothelial turnover is up to hundreds of days. Under angiogenic conditions the turnover time speeds up rapidly to a turnover of under five days, which corresponds with the proliferation of bone marrow cells. This adaptation is of vital importance for the cells to live up to the regenerative demands (Kalluri, 2003).

Other, non-endothelial cells are regulated by VEGF via autocrine control and contribute directly or indirectly to the stimulated processes: monocytes, macrophages, mast cells, dendritic cells, lymphocytes, hematopoietic cells, epithelia, hepatocytes and many others (Breen, 2007).
