Endothelial PGC-1 mediates vascular dysfunction in...
Transcript of Endothelial PGC-1 mediates vascular dysfunction in...
Endothelial PGC 1α mediates Endothelial PGC-1α mediates vascular dysfunction in diabetesvascular dysfunction in diabetes
Reporter: Yaqi Zhouepo te aq ou
Date: 04/14/2014
Outline
I. Introduction
II R h t & R ltII. Research route & Results
III SummaryIII. Summary
Diabetes the Epidemic of the 21st CenturyDiabetes – the Epidemic of the 21st Century1 out of 10 adults will have diabetes in 2035
Number of people with diabetes in different areas by IDF Region, 2013
Diabetes - Endothelial dysfunction
Principle mechanisms responsible for endothelial dysfunction in diabetes.
PGC-1α
PGC‐1α, a transcriptional regulator related to energy metabolism, belongs to a small family of coactivators, comprised of PGC‐1α, PGC‐1β, and the more distant PRC.
PGC-1α & Angiogenesis
PGC-1α
Myocyte
HIF-1 VEGF
PGC 1αPGC-1α
Endothelial cell
Research Route
PGC-1α Endothelial dysfunctionDiabetes
Knock-OutOver-ExpressionMigration
dysfunction
Migration
Transwell assay
Scratch assay Notch signaling
Tube formation
Angiogenesis
1 Di b t i d PGC 1 i i EC1. Diabetes induces PGC-1α expression in ECs
Figure 1.1. Diabetes induces PGC-1α expression in ECs in vivo both in mice and in humans.(A) Relative PGC‐1α mRNA abundance in ECs freshly isolated from mouse models of type 1 (STZ) and type 2 (HFD, ob/ob, and db/db)
diabetes. NC indicates normal chow.( ) l b d l l ll d l d d h l l ll ( ) l d(B) Relative PGC‐1α mRNA abundance in vasculogenic circulating CD34+ cells and cultured endothelial progenitor cells (EPCs) isolated
from patients with diabetes, versus matched normal subjects.
1 Di b t i d PGC 1 i i EC1. Diabetes induces PGC-1α expression in ECs
Figure 1.2. Diabetes, via hyperglycemia, induces PGC-1α expression in ECs.(C) Relative PGC‐1α mRNA abundance in mouse muscle ECs (MMECs) at the indicated times after changing from hyperglycemia to
normal glucose levels.(D) R l ti PGC 1 RNA b d i MMEC i b f 2 d l (2DG) f 24 h(D) Relative PGC‐1α mRNA abundance in MMECs in absence or presence of 2‐deoxyglucose (2DG) for 24 hr.(E) Relative PGC‐1α mRNA abundance in mouse heart ECs (MHECs) 2 and 4 hr after exposing cells to hyperglycemia or
hyperosmolarity (mannitol).
Conclusion Ⅰ
Diabetes, likely at least in part via hyperglycemia, induces PGC-1α expression in ECs in vivo.
Research Route
PGC-1α Endothelial dysfunctionDiabetes
Knock-OutOver-ExpressionMigration
dysfunction
Migration
Transwell assay
Scratch assay Notch signaling
Tube formation
Angiogenesis
2 PGC 1 i hibit d th li l i ti2. PGC-1α inhibits endothelial migration
Figure 2.1. PGC-1α inhibits endothelial migration.(A) Mouse heart ECs (MHECs) were affinity‐purified from STZ‐treated mice and cultured to passage 2
(left). The migrated cells were less than those in control, coincident with increased expression of PGC 1 RNA ( i h )PGC‐1α mRNA (right).
2 PGC 1 i hibit d th li l i ti2. PGC-1α inhibits endothelial migration
Figure 2.2. PGC-1α inhibits endothelial migration.Reduced migration of mouse lung ECs (MLECs) isolated from db/db mice toward S1P (B) and cultured Endothelial progenitor cells(EPCs) isolated from diabetic patients (C). (D) Reduced migration (left) of MHECs isolated from mice that overexpressPGC 1 ( i h ) i ECPGC‐1α (right) in ECs.
2 PGC 1 i hibit d th li l i ti2. PGC-1α inhibits endothelial migration
Figure 2.3 PGC-1α inhibits endothelial migration.(E–G) Stable expression of PGC‐1α inhibits migration of HUVECs in scratch assays (E) and in Transwell assays stimulated by 10 ng/ml VEGF (F) or 100 nM S1P (G).
2 PGC 1 i hibit d th li l i ti2. PGC-1α inhibits endothelial migration
Figure 2.4. PGC-1α inhibits endothelial migration.g g(H) Stable expression of PGC‐1α inhibits the tube‐forming activity of HUVECs.
2 PGC 1 i hibit d th li l i ti2. PGC-1α inhibits endothelial migration
Figure 2.5. PGC-1α inhibits endothelial migration.PGC‐1α inhibits cortical F‐actin (green) polymerization in response to VEGF and S1P (I), mimicking the impaired F‐actin (red) accumulation to the same stimuli in db/db mouse lung ECs (J). Arrowheads denote lamellipodia. Scale bars are 50mm in (I) and 20mm in (J).
2 PGC 1 i hibit d th li l i tiVEGF and S1P act through distinct cell surface receptors but converge on at least two
i ll l i li d h ERK d AKT h E d h li l ll k l l
2. PGC-1α inhibits endothelial migration
intracellular signaling cascades: the ERK and AKT pathways. Endothelial cell cytoskeletalactivation occurs in large part in response to Rac/Akt/eNOS signaling.
Figure 2.6. PGC-1α inhibits endothelial migration.PGC‐1α blocks activation of Akt and eNOS (but not Erk) by VEGF and S1P ([K], with densitometric quantification at 0 and 5 min in[L]) in human endothelial colony‐ forming cells (ECFCs).
Conclusion Ⅱ
PGC-1α inhibits endothelial migration and Akt/eNOSproangiogenic pathway.
Research Route
PGC-1α Endothelial dysfunctionDiabetes
Knock-OutOver-ExpressionMigration
dysfunction
Migration
Transwell assay
Scratch assay Notch signaling
Tube formation
Angiogenesis
Notch signaling pathwayN t h i li i i i l i d f l i hibit f d th li lNotch signaling is increasingly recognized as a powerful inhibitor of endothelial
migration and angiogenesis. The Notch cell surface receptor is sequentially cleaved bymatrix metalloproteinase and γ‐secretase, leading to nuclear localization of the Notchintracellular domain (NICD)intracellular domain (NICD).
3 PGC 1 ti t N t h i li t i hibit d th li l3. PGC-1α activates Notch signaling to inhibit endothelial migration
Figure 3.1. PGC-1α activates Notch signaling both in culture and in vivo.(A) Expression of PGC‐1α in HUVECs activates Notch signaling, as assessed by western blotting of the Notch intracellular
domain (NICD). ( )(B) mRNA expression of known Notch target genes in the endothelial compartment of the mouse skeletal muscle from
EC‐specific PGC‐1α‐overexpressing mice, versus controls.
3 PGC 1 ti t N t h i li t i hibit d th li l3. PGC-1α activates Notch signaling to inhibit endothelial migration
Figure 3.2. PGC-1α activates Notch signaling to inhibit endothelial migration.(C) Inhibition of Notch signaling with DAPT (2.5mM) rescues inhibition of migration in HUVECs retrovirally transduced
with PGC‐1α. DAPT, an inhibitor of γ‐secretase., γ(D) Inhibition of Notch signaling with DAPT (2.5mM) rescues inhibition of capillary formation in mouse aortic explants
from EC‐specific PGC‐1α‐overexpressing mice.
3 PGC 1 ti t N t h i li t i hibit d th li l3. PGC-1α activates Notch signaling to inhibit endothelial migration
Figure 3.3. PGC-1α activates Notch signaling to inhibit endothelial migration.(E) DAPT (2.5 mM) and pan‐MMP inhibitor GM6001 (25mM) equally inhibited the upregulation of Notch downstream
gene mRNA expression in HUVECs retrovirally transduced with PGC‐1α. GM6001, a pan‐MMP inhibitor.g p y , p
3 PGC 1 ti t N t h i li t i hibit d th li l3. PGC-1α activates Notch signaling to inhibit endothelial migration
Figure 3.4. PGC-1α activates Notch signaling to inhibit endothelial migration.(F) Increased mRNA abundance of ADAMTS10 in the MHECs isolated from EC‐specific PGC‐1α transgenic mice, the
endothelial compartment of STZ‐induced diabetic mouse lung, cultured EPCs from diabetic patients, and human p g, p ,dermal microvascular ECs treated with high glucose for 24 hr. ADAMTS10, an ADAM‐like MMP.
3 PGC 1 ti t N t h i li t i hibit d th li l3. PGC-1α activates Notch signaling to inhibit endothelial migration
Figure 3.5. PGC-1α activates Notch signaling to inhibit endothelial migration.(G) Gene knockdown efficiency by lentiviral transduction of HUVECs with short hairpin RNA targeting ADAMTS10.(H and I) Knockdown of ADAMTS10 with shRNA blocked the mRNA expression of Notch target genes (H) and reduced the ( ) p g g ( )inhibition of endothelial migration toward S1P (I) by retroviral overexpression of PGC‐1α in HUVECs.
Conclusion Ⅲ
PGC-1α inhibits endothelial migration at least in part via activation of Notch signaling.
4 L f PGC 1 ti t EC i ti4. Loss of PGC-1α activates EC migration
Figure 4.1. Angiogenic stimuli inhibits endothelial PGC-1α expression.(A) Relative PGC‐1α mRNA abundance in human dermal microvascular ECs stimulated with VEGF (10 ng/mL),
S1P (100 nM), or S‐nitrosoglutathione (GSNO,100mM). GSNO, an NO donor.( ), g ( , ) ,
4 L f PGC 1 ti t EC i ti4. Loss of PGC-1α activates EC migration
Figure 4.2. Loss of PGC-1a activates EC migration.(B and C) Isolated MHECs from PGC‐1α‐/‐ (KO) mice are hypermigratoryin scratch assays (B) and Transwell migration assays (C).(D) Capillary formation of KO (n = 31) and wild‐type (WT) (n = 16) mouse aortic explantsmouse aortic explants.
4 L f PGC 1 ti t EC i ti4. Loss of PGC-1α activates EC migration
Figure 4.3. Loss of PGC-1α inhibits Notch target genes expression.(E) mRNA expression of Notch target genes was depressed in ECs isolated from KO mice, versus wild‐type controls.
4 L f PGC 1 ti t EC i ti4. Loss of PGC-1α activates EC migration
Figure 4.4. High expression of Rac1 in PGC-1α KO ECs.(F) GTP‐Rac1 pull‐down assay in WT and KO MHECs.(G) VEGF (10 ng/ml)‐induced Transwell migration of KO and WT MHECs, with or without DPI (10mM), L‐NAME, or Rac( ) ( g/ ) g , ( ), ,
inhibitor (NSC23766, 30mM).
Conclusion Ⅳ
PGC-1α powerfully inhibits Rac/Akt/eNOS signaling and migration in ECs.
Research Route
PGC-1α Endothelial dysfunctionDiabetes
Knock-OutOver-ExpressionMigration
dysfunction
Migration
Transwell assay
Scratch assay Notch signaling
Tube formation
Angiogenesis
5 E d th li l PGC 1 i hibit i i i i5. Endothelial PGC-1α inhibits angiogenesis in vivo
Figure 5.1. Overexpression of endothelial PGC-1α blunts the formation of new blood vessels.(A) In vivo vasculogenesis assay using mesenchymal progenitor cells and human endothelial colony‐forming cells (ECFCs)
expressing PGC‐1α coinjected nude mice versus control vector. Left, sample appearance of the Matrigel plugs. p g j , p pp g p gMiddle, hematoxylin and eosin staining of plug sections. Right, quantification of vessel density.
5 E d th li l PGC 1 i hibit i i i i5. Endothelial PGC-1α inhibits angiogenesis in vivo
Figure 5.2. Induced expression of PGC-1α in ECs.(B) Schema of Tet‐off and Tet‐on tetracycline‐inducible, endothelial‐specific PGC‐1α transgenic (Tg) mice.(C) mRNA abundance of PGC 1α in freshly isolated lung ECs from Tet on mouse versus control(C) mRNA abundance of PGC‐1α in freshly isolated lung ECs from Tet‐on mouse versus control.
5 E d th li l PGC 1 i hibit i i i i5. Endothelial PGC-1α inhibits angiogenesis in vivo
Figure 5.3. Transgenic expression of PGC-1α inhibits re-endothelialization.Quantification (D) and representative images (E) of re‐endothelialization following wire injury of mouse carotid arteries in Tet‐off transgenic animals.g
5 E d th li l PGC 1 i hibit i i i i5. Endothelial PGC-1α inhibits angiogenesis in vivo
Figure 5.4. Transgenic expression of PGC-1α inhibits wound healing.Quantification (F) and representative images (G) of skin wound healing in Tet‐off transgenic animals.
5 E d th li l PGC 1 i hibit i i i i5. Endothelial PGC-1α inhibits angiogenesis in vivo
Figure 5.5. Transgenic expression of PGC-1α inhibits the rate of blood flow recovery in hindlimb ischemia models.Quantification of blood flow recovery (H) and necrotic toes (I) after induced hindlimb ischemia in Tet‐on animals.
Conclusion Ⅴ
Expression of PGC-1α in ECs in vivo mimics multiple aspects of diabetic EC dysfunction.
6 Loss of endothelial PGC-1α rescues diabetic endothelial6. Loss of endothelial PGC-1α rescues diabetic endothelial dysfunction
Figure 6.1. Loss of endothelial PGC-1α accelerates wound healing in diabetic mice.(A) Skin wound healing in endothelial‐specific PGC‐1α knockout (EC‐KO) and control mice, either nondiabetic or rendered
diabetic by treatment with STZ.(B) Ski d h li i EC KO d l i h f d HFD f 12 h(B) Skin wound healing in EC‐KO and control mice that were fed HFD for 12 months.
6 Loss of endothelial PGC-1α rescues diabetic endothelial6. Loss of endothelial PGC-1α rescues diabetic endothelial dysfunction
Figure 6.2. Loss of endothelial PGC-1α recovers blood flow and decreases the number of necrotic toes.Recovery of blood flow after surgical induction of hindlimb ischemia in type 1 diabetic EC‐KO mice: (C) representative day 14 laser Doppler perfusion images, (D) quantification of blood perfusion recovery in the ischemic limbs, (E) number f i i h i h i f d 28 d (F) i f di b i i h i li bof necrotic toes in the ischemic foot at day 28, and (F) representative appearance of diabetic mouse ischemic limbs at
day 14.
6 Loss of endothelial PGC-1α rescues diabetic endothelial6. Loss of endothelial PGC-1α rescues diabetic endothelial dysfunction
Figure 6.3. Loss of endothelial PGC-1α recovers blood flow and decreases the number of necrotic toes.Recovery of blood flow after hind limb ischemia in type 2 diabetic EC‐KO mice: (G) quantification of blood perfusion recovery in the ischemic limbs and (H) number of necrotic toes in the ischemic foot at day 21.
Conclusion Ⅵ
Deletion of PGC-1α in ECs in large part rescues numerous aspects of endothelial dysfunction in diabetes.
Summary
Significance
Supporting the notion that induction of PGC‐1α in ECs mediates Supporting the notion that induction of PGC 1α in ECs mediates endothelial dysfunction and vascular complications in diabetes.
Identifying an important relationship between angiogenesis and Identifying an important relationship between angiogenesis and a central regulator of metabolism.
Revealing the opposing effects of PGC‐1α in different cells which Revealing the opposing effects of PGC 1α in different cells which stresses the need for caution in therapies aimed at PGC‐1α in this and other contexts.