**2. Hypoxia inducible factors**

Hypoxia Inducible Factors (HIF) are transcriptional factors that were discovered in mammalian cells under conditions of low oxygen, and appear to have a fundamental role in the cellular and systematic response to hypoxia, via the regulation of metabolism, cellular cycle, angiogenesis and apoptosis. HIFs are heterodimers constituted from the HIFa (1, 2 or 3) and HIFb or ARNT.

HIFa subunit is found in the cytoplasm and is transported in the nuclei in order to form HIF with the other subunit ARNT, which is expressed regularly in the nuclei, and achieve its transcriptional role. It has been found that transcriptional action of HIFa is achieved via bHLH (basic helix-loop-helix)-PAS domain in N-terminal. For this action the recruitment of

Placental Angiogenesis and Fetal Growth Restriction 183

genes that play role in angiogenesis, cellular cycle and metabolism [Semenza et al., 2000]. Similar function appears also for the HIF-2, while for HIF-3 the existing data is insufficient. The regulation of these factors is depending on the partial pressure of oxygen, a fact that makes them immediately connected with hypoxic conditions in the feto-placental circulation

Fig. 1. Graphical representation of the HIF pathway during normoxic and hypoxic

forms a dimer with ARNT, which then binds to DNA, leading to gene transcription

activation [Gourvas et al., 2010].

transcription [Lando et al., 2002].

conditions. In normoxia (grey arrows) PHD hydroxylates HIF-a, which then binds to vHL, leading to its proteosomal degradation. In hypoxia (black arrows) HIF-a enters the nucleus,

Regulation of HIFs appears to relate not only with the induction/suspension of their expression, but also with their degradation. Thus, in physiologic conditions the a- subunits of HIF are degradated with a mechanism that includes factors PHD-1, 2 and 3 (prolyl hydroxylation domain) and VHL. They are responsible for the hydroxylation and degradation of HIFa, so that HIFa cannot activate ARNT in the nuclei. In a second phase one more hydroxylation can contribute in the suspension of HIF, which becomes in asn803 and suspends the interaction with co-activators p300/CBP, which is essential for the induction of

such as preeclampsia or FGR [Smith et al., 2001].

co-activators p300 and CBP is needed. While HIF-1 and HIF-2 have similar structure and action, it appears that HIF-3 presents suspensive action in the hypoxia, which has not however been confirmed [Kiichi et al., 2006].

HIFa under normoxic conditions is hydroxylated in two Prolyl domains (Pro 402 and Pro 564) by their Prolyl Hydroxylation Domains (PHD-1,2 and 3), which induces the reaction with the von Hippel-Lindau (VHL) forming a dimmer that is proteosomical degradated. It appears that VHL is downregulated in hypoxia, consequently releasing the HIFa that at the same time is upregulated. PHD utilizes Ο2 as a substrate with a Km that is slightly above atmospheric concentration. The enzymatic activity is modulated by changes in Ο<sup>2</sup> concentration under physiological conditions. This regulation appears to be common for all three HIFa factors [Min et al., 2002].

Deductively, the HIFa factors are upregulated by hypoxia, while the ARNT is stably expressed and in order to they carry out their transcriptional role, they should form a heterodimmer in the nuclei. Under normal conditions, while HIFs continue to be expressed, VHL and PHD-1, 2 and 3 are upregulated in order to complete protesomical degradation of HIFs. The existing data for HIF-1a and HIF-2a show a regulating role in angiogenesis, metabolism, cellular cycle and apoptosis; for HIF-3a however there is still lack of evidence regarding his precise role [Makino et al., 2001]. On the contrary, factors PHD-1, 2 and 3 appear to play all similar roles in the degradation of HIFa subunits [Salceda et al., 1997; Maxwell et al., 1999]. Important examples of factors that are regulated by HIF-1 are VEGF, PLGF, Flt-1 and Angiopoietin-2 regarding arterial destabilization and increased vascular permeability, MMPs, Angiopoietin-1, MCP-1 and PDGF regarding migration and proliferation of endothelial cells etc. Figure 1 represents the HIF pathway during normoxic and hypoxic conditions. regulation hypoxic the

#### **2.1 Hypoxia inducible factors in placenta**

It is already known that one of the most important processes during gestation is the physiologic development of feto-placental unit that begins with the cytotrophoblast invasion in the endometrium and is completed with the creation of chorionic villi [Huppertz et al., 2007]. Initially the cytotrophoblast environment can be characterized as hypoxic, but after the completion of the conjunction with the spiral vessels there is a passage to physiologic oxygenation. During this process, structural changes are required for both the epithelium of endometrium and the endothelium and walls of the spiral arteries. The wall of vessels that is found to the side of fetus is replaced from trophoblasts. Cytotrophoblasts in this process turn from an expressive pattern of molecules of epithelial specification in an expressive pattern of adherence molecules that is more of endothelium cells differentiation. Many of these molecules are regulated by HIF. The cytotrophoblast tends to proliferate in hypoxia but to differentiate in physiological oxygenation, and these data show that cytotrophoblast activity depends on the presence of oxygen.

Results from studies in animal models have shown that at the initial stages of pregnancy where PO2 is decreased, an increased action of regulating factors exists, mainly from HIF-1, HIF-2 and HIF-3. HIF-1 comprises a basic response of a cell to the hypoxic conditions. It is constituted by two subunits, HIF-1a and HIF-1b (ARNT). HIF-1a forms a heterodimmer with the HIF-1b and HIF-1, acts on the cell nucleus and regulates the expression of many

co-activators p300 and CBP is needed. While HIF-1 and HIF-2 have similar structure and action, it appears that HIF-3 presents suspensive action in the hypoxia, which has not

HIFa under normoxic conditions is hydroxylated in two Prolyl domains (Pro 402 and Pro 564) by their Prolyl Hydroxylation Domains (PHD-1,2 and 3), which induces the reaction with the von Hippel-Lindau (VHL) forming a dimmer that is proteosomical degradated. It appears that VHL is downregulated in hypoxia, consequently releasing the HIFa that at the same time is upregulated. PHD utilizes Ο2 as a substrate with a Km that is slightly above atmospheric concentration. The enzymatic activity is modulated by changes in Ο<sup>2</sup> concentration under physiological conditions. This regulation appears to be common for all

Deductively, the HIFa factors are upregulated by hypoxia, while the ARNT is stably expressed and in order to they carry out their transcriptional role, they should form a heterodimmer in the nuclei. Under normal conditions, while HIFs continue to be expressed, VHL and PHD-1, 2 and 3 are upregulated in order to complete protesomical degradation of HIFs. The existing data for HIF-1a and HIF-2a show a regulating role in angiogenesis, metabolism, cellular cycle and apoptosis; for HIF-3a however there is still lack of evidence regarding his precise role [Makino et al., 2001]. On the contrary, factors PHD-1, 2 and 3 appear to play all similar roles in the degradation of HIFa subunits [Salceda et al., 1997; Maxwell et al., 1999]. Important examples of factors that are regulated by HIF-1 are VEGF, PLGF, Flt-1 and Angiopoietin-2 regarding arterial destabilization and increased vascular permeability, MMPs, Angiopoietin-1, MCP-1 and PDGF regarding migration and proliferation of endothelial cells etc. Figure 1 represents the HIF pathway during normoxic

It is already known that one of the most important processes during gestation is the physiologic development of feto-placental unit that begins with the cytotrophoblast invasion in the endometrium and is completed with the creation of chorionic villi [Huppertz et al., 2007]. Initially the cytotrophoblast environment can be characterized as hypoxic, but after the completion of the conjunction with the spiral vessels there is a passage to physiologic oxygenation. During this process, structural changes are required for both the epithelium of endometrium and the endothelium and walls of the spiral arteries. The wall of vessels that is found to the side of fetus is replaced from trophoblasts. Cytotrophoblasts in this process turn from an expressive pattern of molecules of epithelial specification in an expressive pattern of adherence molecules that is more of endothelium cells differentiation. Many of these molecules are regulated by HIF. The cytotrophoblast tends to proliferate in hypoxia but to differentiate in physiological oxygenation, and these data show that

Results from studies in animal models have shown that at the initial stages of pregnancy where PO2 is decreased, an increased action of regulating factors exists, mainly from HIF-1, HIF-2 and HIF-3. HIF-1 comprises a basic response of a cell to the hypoxic conditions. It is constituted by two subunits, HIF-1a and HIF-1b (ARNT). HIF-1a forms a heterodimmer with the HIF-1b and HIF-1, acts on the cell nucleus and regulates the expression of many

however been confirmed [Kiichi et al., 2006].

three HIFa factors [Min et al., 2002].

and hypoxic conditions.

**2.1 Hypoxia inducible factors in placenta** 

cytotrophoblast activity depends on the presence of oxygen.

genes that play role in angiogenesis, cellular cycle and metabolism [Semenza et al., 2000]. Similar function appears also for the HIF-2, while for HIF-3 the existing data is insufficient. The regulation of these factors is depending on the partial pressure of oxygen, a fact that makes them immediately connected with hypoxic conditions in the feto-placental circulation such as preeclampsia or FGR [Smith et al., 2001].

Fig. 1. Graphical representation of the HIF pathway during normoxic and hypoxic conditions. In normoxia (grey arrows) PHD hydroxylates HIF-a, which then binds to vHL, leading to its proteosomal degradation. In hypoxia (black arrows) HIF-a enters the nucleus, forms a dimer with ARNT, which then binds to DNA, leading to gene transcription activation [Gourvas et al., 2010].

Regulation of HIFs appears to relate not only with the induction/suspension of their expression, but also with their degradation. Thus, in physiologic conditions the a- subunits of HIF are degradated with a mechanism that includes factors PHD-1, 2 and 3 (prolyl hydroxylation domain) and VHL. They are responsible for the hydroxylation and degradation of HIFa, so that HIFa cannot activate ARNT in the nuclei. In a second phase one more hydroxylation can contribute in the suspension of HIF, which becomes in asn803 and suspends the interaction with co-activators p300/CBP, which is essential for the induction of transcription [Lando et al., 2002].

Placental Angiogenesis and Fetal Growth Restriction 185

Various decidual cell types are capable of producing angiogenic factors. We recently showed the production of PlGF, KDR, Flt-1, Ang-2, and TIE-2 by endothelial cells and extravillous trophoblasts. Decidual stromal cells, glandular epithelium, and perivascular smooth muscle cells were found to produce all studied angiogenic factors [Plaisier et al., 2007]. Uterine natural killer cells are also abundantly present in first-trimester decidua and

A successful pregnancy outcome depends on the proper development of the fetoplacental vasculature in the villous core, which begins with the infiltration of cytotrophoblast in the endometrium and is completed in conjunction with the spiral arteries. It is widely accepted that shallow trophoblast invasion can lead to fetal hypoxia and impaired growth. The proper and timely proliferation and differentiation of the villous cytotrophoblast stem cells, which are controlled by hypoxia, are crucial for adequate placentation and initiation of angiogenetic pathways. Numerous factors are thought to play a role in normal vascular adaptation to implantation. There is strong evidence that abnormal levels of angiogenic and antiangiogenic growth factors could in part be responsible for the pathophysiology

Ahmed AS, Li XF, Dunk CE, Whittle MJ, Rollason T. Colocalisation of vascular endothelial

Ahmed A, Dunk C, Ahmad S, Khaliq A. Regulation of placental vascular endothelial growth

Ahmed A, Perkins J: Angiogenesis and intrauterine growth restriction. Baillieres Best Pract

Asahara T, Chen D, Takahashi T, et al. Tie2 receptor ligands, angiopoietin-1 and

Benirschke K, Kaufmann P (Eds). Pathology of Human Placenta. London: Springer-Verlag

Cetin I, Foidart JM, Miozzo M, et al. Fetal growth restriction: a workshop report. Placenta

Chung J, Song Y, Wang Y, Magness RR, Zheng J. Differential expression of vascular

Demir R, Kaufmann P, Castellucci M, Erbengi T, Kotowski A. Fetal vasculogenesis and angiogenesis in human placental villi. Acta Anat (Basel) 1989;136:190–203 Demir R, Seval Y, Huppertz B. Vasculogenesis and angiogenesis in the early human

Flamme I, Frolich T, Risau W. Molecular mechanisms of vasculogenesis and embryonic

growth factor and its flt-1 receptor in human placenta. Growth Factors 1995;12:235-

factor (VEGF) and placenta growth factor (PlGF) and soluble flt-1 by oxygen. A

angiopoietin-2, modulate VEGF-induced postnatal neovascularization. *Circ Res.* 

endothelial growth factor (VEGF), endocrine gland derived-VEGF, and VEGF receptors in human placentas from normal and preeclamptic pregnancies. *J Clin* 

are known to produce PlGF, VEGF, Ang-1, and Ang-2.

associated with pregnancies complicated by FGR.

Review. Troph Res 2000;14:16-24.

*Endocrinol Metab* 2004;89:2484-2490.

placenta. Acta Histochem 2007;109:257-265.

angiogenesis. J Cell Physiol 1997;173:206–210 Folkman J, Klagsbrun M. Angiogenic factors. Science 1987;233:442–447

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**4. Conclusion** 

**5. References** 

243.

1995

1998;83:233-240

2004;25:753-757.

It is obvious that the regulation of HIF expression and function is achieved through a number of factors, such as PO2, VHL and PHD-1, 2 and 3. Consequently, a measurement of expression only of HIF in conditions with decreased supply of oxygen may lead to false conclusions, especially by considering that their regulation is also posttranscriptional through the action of PHD and VHL. Later during gestation, when fetal-placental unit provides satisfactory quantities of O2, HIFs are downregulated by decreased expression or degradation. In pathological situations, however, it is probable that this model is disturbed and the maintenance of decreased oxygenation affects the regulating action of these factors. Thus exists a change/imbalance in the expression of genes related with processes such as angiogenesis (VEGF, PLGF, PDGF, EPO, NOS2, FLT1 etc), metabolism (aldolase, hexokinase, pyrouvic kinase, lactic dehydrogonase etc) and cellular cycle (IGF, p21, p35srj etc). These changes may produce clinical signs and symptoms of FGR or preeclampsia or of both of them. of increased
