80
merize with HIFβ (ARNT) and form a transcriptional complex which regulates the
transcription of numerous genes [159]. Stabilization of HIFα in the cell is controlled
by two main O 2 sensing proteins, prolyl hydroxylase domain (PHD) and factorinhibiting HIFα (FIH), which belong to the previously mentioned biosynthetic sensors category. Three isoforms of PHDs are present in all mammals [28]. Specific
proline residues on the oxygen-dependent domain of HIFα are hydroxylated by
PHDs at separate hydroxylation sites, leading to HIFα degradation. The activity of
PHDs in the cytoplasm is controlled by various O 2 -dependent molecular events and,
directly, by the concentration of the O 2 molecule [68]. All three PHDs remain partially active in normoxia. PHD activity is expected to be very sensitive to small
changes in cytoplasmic O 2 levels since K m , the Michaelis-Menten parameter for the
activation of PHDs, is approximately 230–250 μM, which is much higher than physiological oxygen concentration (approximately 60 μM) [98]. Besides, mitochondria
are also involved in the PHD activation process through their consumption of O 2 ,
regulation of reactive oxygen species (ROS), and production of nitric oxide (NO).
While the stabilization of HIFα depends on PHD activity, the expression of HIFα is
controlled by FIHs. Therefore, when O 2 levels are lowered, both the stabilization and
transactivation of HIFα increase, resulting in several angiogenic responses that will
be discussed in the following section.
NO and ROS not only contribute to the HIFα stabilization process, but they also
have several direct effects on vascular cells and blood vessels. A number of studies
have shown that NO induces angiogenesis, hyperpermeability, and vasodilation
[73]. Moreover, NO also perturbs EC respiration through the inhibition of cytochrome c oxidase, which causes lower mitochondrial O 2 consumption [118].
Mitochondrial ROS are also increased as a consequence of electron transport chain
inhibition, which then contributes to the deactivation of PHDs via oxidizing cofactor Fe (II) and helps to stabilize HIFα. ROS production, in respect to hypoxia, is
proportional to the concentrations of intracellular O 2 and electron donors. Under
hypoxia, the amount of O 2 required to form superoxides is decreased, whereas the
concentration of the electron donors increases as a consequence of the reduction in
the proximal electron transport chain. Therefore, ROS production can change in
both manners, depending on the variations in these molecules’ concentrations [233].
Ushia-Fukari et al. [227] showed that ROS influence the expression of surface adhesion molecules of ECs and stimulate EC proliferation and vessel permeability.
Moreover, the hypoxia-induced decrease in ROS production leads to the inhibition
of K
+
channels of pulmonary artery smooth muscle cells (SMCs), whereas an
increase in ROS production leads to intracellular Ca
+
release from ryanodinesensitive stores [233]. Another molecular path found between mitochondrial energy
generation and K
+
channel inhibition occurs through AMP kinases. The energy of
the cell is generated by the conversion of ADP to one molecule of ATP and
AMP. Hence, AMP kinase becomes highly dependent on the ADP/ATP ratio, which
is very sensitive to changes in cytoplasmic O 2 concentrations. AMP kinases were
shown to inhibit K
+
channels through the regulation of Ca
+
release in pulmonary
arterial SMCs and also to induce cellular survival in tumor cells when exposed to
severe hypoxia [63, 171].
M. R. Blatchley et al.
merize with HIFβ (ARNT) and form a transcriptional complex which regulates the
transcription of numerous genes [159]. Stabilization of HIFα in the cell is controlled
by two main O 2 sensing proteins, prolyl hydroxylase domain (PHD) and factorinhibiting HIFα (FIH), which belong to the previously mentioned biosynthetic sensors category. Three isoforms of PHDs are present in all mammals [28]. Specific
proline residues on the oxygen-dependent domain of HIFα are hydroxylated by
PHDs at separate hydroxylation sites, leading to HIFα degradation. The activity of
PHDs in the cytoplasm is controlled by various O 2 -dependent molecular events and,
directly, by the concentration of the O 2 molecule [68]. All three PHDs remain partially active in normoxia. PHD activity is expected to be very sensitive to small
changes in cytoplasmic O 2 levels since K m , the Michaelis-Menten parameter for the
activation of PHDs, is approximately 230–250 μM, which is much higher than physiological oxygen concentration (approximately 60 μM) [98]. Besides, mitochondria
are also involved in the PHD activation process through their consumption of O 2 ,
regulation of reactive oxygen species (ROS), and production of nitric oxide (NO).
While the stabilization of HIFα depends on PHD activity, the expression of HIFα is
controlled by FIHs. Therefore, when O 2 levels are lowered, both the stabilization and
transactivation of HIFα increase, resulting in several angiogenic responses that will
be discussed in the following section.
NO and ROS not only contribute to the HIFα stabilization process, but they also
have several direct effects on vascular cells and blood vessels. A number of studies
have shown that NO induces angiogenesis, hyperpermeability, and vasodilation
[73]. Moreover, NO also perturbs EC respiration through the inhibition of cytochrome c oxidase, which causes lower mitochondrial O 2 consumption [118].
Mitochondrial ROS are also increased as a consequence of electron transport chain
inhibition, which then contributes to the deactivation of PHDs via oxidizing cofactor Fe (II) and helps to stabilize HIFα. ROS production, in respect to hypoxia, is
proportional to the concentrations of intracellular O 2 and electron donors. Under
hypoxia, the amount of O 2 required to form superoxides is decreased, whereas the
concentration of the electron donors increases as a consequence of the reduction in
the proximal electron transport chain. Therefore, ROS production can change in
both manners, depending on the variations in these molecules’ concentrations [233].
Ushia-Fukari et al. [227] showed that ROS influence the expression of surface adhesion molecules of ECs and stimulate EC proliferation and vessel permeability.
Moreover, the hypoxia-induced decrease in ROS production leads to the inhibition
of K
+
channels of pulmonary artery smooth muscle cells (SMCs), whereas an
increase in ROS production leads to intracellular Ca
+
release from ryanodinesensitive stores [233]. Another molecular path found between mitochondrial energy
generation and K
+
channel inhibition occurs through AMP kinases. The energy of
the cell is generated by the conversion of ADP to one molecule of ATP and
AMP. Hence, AMP kinase becomes highly dependent on the ADP/ATP ratio, which
is very sensitive to changes in cytoplasmic O 2 concentrations. AMP kinases were
shown to inhibit K
+
channels through the regulation of Ca
+
release in pulmonary
arterial SMCs and also to induce cellular survival in tumor cells when exposed to
severe hypoxia [63, 171].
M. R. Blatchley et al.