83
vessels formed, which then led to tissue edema and inflammation [184]. On the
other hand, overexpressing both VEGF and ANG-1, which is important for maintaining vascular integrity, has been shown to induce hypervascularity without
imperfections in mice [218]. ANG-2 is responsible for EC apoptosis and vascular
regression in the absence of VEGF, whereas, when combined with VEGF expression, it enhances angiogenic responses by destabilizing the blood vessels [100,
101]. More recently, ANG-4 was shown to function similarly to ANG-1 and to
induce angiogenesis by binding the ANG receptor TIE-2, which is also upregulated
by HIF1α [241]. We have recently shown that VEGF and ANG-2 genes are upregulated in hypoxic (1% O 2 ) cultures of EPCs and HUVECs [1], and the fold differences in upregulation levels of VEGF and ANG-2 in EPCs were shown to vary
during the 3-day exposure period (Fig. 4.1b), where no significant change was
observed for HUVECs (Fig. 4.1c). How hypoxia affects the regulation of these
angiogenic genes depends on the cell type; for instance, VEGF is upregulated in
ECs, SMCs, cardiac fibroblasts, and myocardiocytes, whereas ANG-2 is induced
only in ECs [159]. Therefore, from a tissue engineering perspective, co-culturing of
different cell types under controlled hypoxic conditions should be considered, since
a combination of hypoxia- induced angiogenic proteins is required to obtain vascular formation without excessive permeability.
Cell Death and Survival
Hypoxia influences the proliferation and viability of many cell types [1, 69, 172,
245]. The wide spectrum of HIF1α-dependent genes also includes proapoptotic and
prosurvival genes. BH3-only proapoptotic genes, a subfamily of BCL-2 that
includes BNIP3, BNIP3L, NOXA, RTP801, and HGTP-P, are directly activated by
HIF1α [234]. Although these genes play important roles in cellular apoptosis, a
growing body of evidence suggests that hypoxia mediates cellular survival in many
cell types [160, 172, 245]. Programmed cell death is, of course, a very critical step
for cells and is most likely taken only after all possible survival mechanisms have
been exhausted. One of these mechanisms, autophagy, is a cellular catabolic process where cytoplasmic organelles are degraded to provide ATP generation in nutrient deprivation. Hypoxia was found to induce mitochondrial autophagy via both
HIF1α- dependent and HIF1α-independent pathways [172, 245]. Small interfering
RNA silencing of BNIP3 and BNIP3L together suppresses autophagy to a greater
extent than silencing only one of them at a time [20]. Zhang et al. have shown that
mitochondrial autophagy is induced by HIF1α-dependent upregulation of BNIP3
incorporated into the constitutive expression of BECLIN-1 and ATG-5 [245]. On
the other hand, the neuron-derived orphan receptor (NOR-1), which is overexpressed in ECs exposed to hypoxia, mediates cellular survival as a downstream
effector of HIF1α signaling [160]. CD105, one of the EC markers also shown to
play a role in cellular survival, is significantly upregulated under hypoxia [146]. In
vivo studies of rats subjected to hypoxia also found the induction of mitochondrial
autophagy by overexpression of BNIP3 [17]. In addition, Papandreou et al. propose
4 Hypoxia and Matrix Manipulation for Vascular Engineering
vessels formed, which then led to tissue edema and inflammation [184]. On the
other hand, overexpressing both VEGF and ANG-1, which is important for maintaining vascular integrity, has been shown to induce hypervascularity without
imperfections in mice [218]. ANG-2 is responsible for EC apoptosis and vascular
regression in the absence of VEGF, whereas, when combined with VEGF expression, it enhances angiogenic responses by destabilizing the blood vessels [100,
101]. More recently, ANG-4 was shown to function similarly to ANG-1 and to
induce angiogenesis by binding the ANG receptor TIE-2, which is also upregulated
by HIF1α [241]. We have recently shown that VEGF and ANG-2 genes are upregulated in hypoxic (1% O 2 ) cultures of EPCs and HUVECs [1], and the fold differences in upregulation levels of VEGF and ANG-2 in EPCs were shown to vary
during the 3-day exposure period (Fig. 4.1b), where no significant change was
observed for HUVECs (Fig. 4.1c). How hypoxia affects the regulation of these
angiogenic genes depends on the cell type; for instance, VEGF is upregulated in
ECs, SMCs, cardiac fibroblasts, and myocardiocytes, whereas ANG-2 is induced
only in ECs [159]. Therefore, from a tissue engineering perspective, co-culturing of
different cell types under controlled hypoxic conditions should be considered, since
a combination of hypoxia- induced angiogenic proteins is required to obtain vascular formation without excessive permeability.
Cell Death and Survival
Hypoxia influences the proliferation and viability of many cell types [1, 69, 172,
245]. The wide spectrum of HIF1α-dependent genes also includes proapoptotic and
prosurvival genes. BH3-only proapoptotic genes, a subfamily of BCL-2 that
includes BNIP3, BNIP3L, NOXA, RTP801, and HGTP-P, are directly activated by
HIF1α [234]. Although these genes play important roles in cellular apoptosis, a
growing body of evidence suggests that hypoxia mediates cellular survival in many
cell types [160, 172, 245]. Programmed cell death is, of course, a very critical step
for cells and is most likely taken only after all possible survival mechanisms have
been exhausted. One of these mechanisms, autophagy, is a cellular catabolic process where cytoplasmic organelles are degraded to provide ATP generation in nutrient deprivation. Hypoxia was found to induce mitochondrial autophagy via both
HIF1α- dependent and HIF1α-independent pathways [172, 245]. Small interfering
RNA silencing of BNIP3 and BNIP3L together suppresses autophagy to a greater
extent than silencing only one of them at a time [20]. Zhang et al. have shown that
mitochondrial autophagy is induced by HIF1α-dependent upregulation of BNIP3
incorporated into the constitutive expression of BECLIN-1 and ATG-5 [245]. On
the other hand, the neuron-derived orphan receptor (NOR-1), which is overexpressed in ECs exposed to hypoxia, mediates cellular survival as a downstream
effector of HIF1α signaling [160]. CD105, one of the EC markers also shown to
play a role in cellular survival, is significantly upregulated under hypoxia [146]. In
vivo studies of rats subjected to hypoxia also found the induction of mitochondrial
autophagy by overexpression of BNIP3 [17]. In addition, Papandreou et al. propose
4 Hypoxia and Matrix Manipulation for Vascular Engineering