**3. Hypoxia: Master and commander of vascular regeneration**

The lack of oxygen immediately threatens the organism's integrity. Few minutes without oxygen supply lead to irreparable damages in the affected organs. The oxygen sensing and the quick and efficient induction of regulative measures are among the most sensitive and finetuned processes in physiology.

#### **3.1. Physiology**

Proliferating endothelial cells are capable of forming three-dimensional structures as tubes

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‐

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

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

The lack of oxygen immediately threatens the organism's integrity. Few minutes without oxygen supply lead to irreparable damages in the affected organs. The oxygen sensing and the

and loops, the structural fundament of a functioning circulation.

for the cells to live up to the regenerative demands (Kalluri, 2003).

**3. Hypoxia: Master and commander of vascular regeneration**

tive process (Pandya, Dhalla et al., 2006).

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

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(Breen, 2007).

Hypoxia is one of the most potent inductors of angiogenesis. Hypoxia is defined by a deficiency of oxygen that can concern the whole organism or parts of it.

The standard is age dependent and varies from 80-100 mmHg, the formula is paO2=102-(age in years x 0.33).

A disturbance of the oxygen haemostasis can be caused on different levels: the partial pressure of the tidal air, the gaseous exchange in the lung or the peripheral tissues or the binding capacity of the erythrocytes.

One has to differentiate different forms of hypoxia: the hypoxic hypoxia refers to a lack of oxygen caused among others reasons by a low partial pressure in heights or by the inability of the lung tissue to perform the necessary gaseous exchange.

The anaemic hypoxia reposes on a reduced capacity of oxygen transport in the blood, e.g. caused by a reduced content of haemoglobin or a carbon monoxide poisoning.

The ischemic hypoxia is caused by a disturbed perfusion of single organs, e.g. due to an embolic insult.

In histotoxic hypoxia the concerned cells are not able to exploit the present oxygen. It is observed in cyanide or alcohol intoxication.

The pathologies resulting from hypoxia are various: on cellular level an alteration of oxygen tension can lead to endothelial changes, among others.

Systemically persisting hypoxia results in pulmonary hypertension. The aim of the increased perfusion of the pulmonary vessels is an optimal oxygen profit during the gaseous exchange.

On bio molecular level hypoxia interferes with gene expression; via oxygen sensing molecules and their downstream signalling cascade the transcription of genes is promoted that induce an enhanced haematopoiesis and angio- and vasculogenesis.

#### **3.2. Oxygen sensing**

Cellular mechanisms of oxygen regulation concern the aerobe glycolysis, the arrest of the cell cycle end the initiation of apoptosis.

Systemic regulation includes the release of erythropoietin from the kidneys, hyperventilation and finally angiogenesis.

The interesting question is: which molecule represents the sensor of a low intracellular oxygen tension?

### **3.3. HIF-1 alpha**

Hypoxia inducible factor 1 alpha (HIF-1 alpha) is the key regulator of cellular and systemic oxygen haemostasis. It has been described in 1995 for the first time by Wang et al. (Wang, Jiang, et al., 1995)

HIF-1 alpha consists of two subunits: the alpha subunit is the virtual oxygen sensor. It is O2 sensitive and very unstable. In presence of oxygen, the alpha subunit is not detectable. Under normoxic conditions a quick ubiquination and immediate proteosomic degradation is observed. Oxygen-dependent enzymes, the prolyl-hydroxylases (PHDs) bind oxygen and couple it to HIF1-alpha. Von Hippel Lindau (VHL) protein attacks this complex and initiates its degradation.

The necessary co-factors for the degradation are oxygen and iron. Under hypoxic conditions HIF-1 alpha cannot be degraded for the lack of the co-factor oxygen and accumulates. The molecules reach the nucleus where the come in contact with the HIF-1 beta subunit.

The beta subunit, the so-called aryl hydrocarbon receptor nuclear translocator, ARNT, is expressed in the nucleus constantly.

Triggered by hypoxia there is a dimerisation of alpha and beta subunit that finally leads to the activation of target genes via binding to so-called hypoxia responsive elements (HREs) (Semenza, 2001).

**Figure 2.** Oxygen sensing (Zagórska, Dulak et al., 2004)

Thus HIF-1 transmits the gene activation that is initiated by the existing hypoxia; many genes – directly or indirectly regulated by hypoxia - are involved in the unleashed cascade that basically leads to cell differentiation, migration and glycolysis. Next to the famous players VEGF, Flk-1 and Flt-1, EPO, LDH-A, platelet-derived growth factor-ß (PDGF-ß) or basic fibroblast growth factor (bFGF) are involved.

#### **3.4. Target genes**

**3.3. HIF-1 alpha**

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et al., 1995)

its degradation.

(Semenza, 2001).

expressed in the nucleus constantly.

**Figure 2.** Oxygen sensing (Zagórska, Dulak et al., 2004)

Hypoxia inducible factor 1 alpha (HIF-1 alpha) is the key regulator of cellular and systemic oxygen haemostasis. It has been described in 1995 for the first time by Wang et al. (Wang, Jiang,

HIF-1 alpha consists of two subunits: the alpha subunit is the virtual oxygen sensor. It is O2 sensitive and very unstable. In presence of oxygen, the alpha subunit is not detectable. Under normoxic conditions a quick ubiquination and immediate proteosomic degradation is observed. Oxygen-dependent enzymes, the prolyl-hydroxylases (PHDs) bind oxygen and couple it to HIF1-alpha. Von Hippel Lindau (VHL) protein attacks this complex and initiates

The necessary co-factors for the degradation are oxygen and iron. Under hypoxic conditions HIF-1 alpha cannot be degraded for the lack of the co-factor oxygen and accumulates. The

The beta subunit, the so-called aryl hydrocarbon receptor nuclear translocator, ARNT, is

Triggered by hypoxia there is a dimerisation of alpha and beta subunit that finally leads to the activation of target genes via binding to so-called hypoxia responsive elements (HREs)

Thus HIF-1 transmits the gene activation that is initiated by the existing hypoxia; many genes – directly or indirectly regulated by hypoxia - are involved in the unleashed cascade that

molecules reach the nucleus where the come in contact with the HIF-1 beta subunit.

A plethora of target genes are governed by hypoxia via HIF-1. The following tables summarize the most relevant genes according to their function in the context of angio- and vasculogenesis


**Table 1.** Extracelullar targets


(Metzen, Ratcliffe et al, 2004, Marxsen, Stengel et al., 2004)

**Table 2.** Intracellular targets

#### **3.5. Clinical relevance**

The functionality of HIF-1 alpha, its capacity to transmit the need for oxygen and therefore for more vasculature has been the basic idea of innovative therapeutic concepts.

Recently several drugs have been developed which act as selective HIF prolyl-hydroxylase inhibitors; the inhibited degradation of HIF-1α persuades the system of a severe lack of oxygen and leads to an initiation of counter-measures.

By inhibiting HIF prolyl-hydroxylase, the activity of HIF-1 alpha in the bloodstream is prolonged, which results in an increase in endogenous production of erythropoietin (Bruegge, Jelkmann et al., 2007).

HIF activity is involved in angiogenesis required for cancer tumour growth, so HIF inhibitors are under investigation for anti-cancer effects (Semenza, 2006).

In addition, there have been observations that suggest that HIF pathway is not only a pivotal inductor of neo-angiogenesis but also is relevant in questions of bone regeneration for example in fracture repair.

The mechanism behind this hypothesis postulates the ability of osteoblasts to instrumentalize HIF-1 alpha as oxygen sensor and the corresponding signalling cascade to improve angio- and osteogenesis concurrently; the molecular interconnection is not finally elucidated. A dynamic crosstalk between osteoblasts and endothelial progenitors is assumed.

Therefore the application of HIF activators might improve bone healing by optimizing the angiogenic properties of the wounded bone but more important by inducing bone regeneration itself. Encouraging observations have been made in mouse fracture models where an overexpression of HIF and VEGF in long bones of mice results in pronounced vascularisation. Even a separate cultivation of the special osteoblasts without the corre‐ sponding endothelial cells does not affect their proliferation and differentiation (Wan, Gilbert et al., 2008, Wang, Wan et al, 2007).
