1) Endothelin-1 (ET-1) promotes vascular endothelial growth factor (VEGF) expression and angiogenesis in human chondrosarcoma cells. ET-1 increases VEGF levels through the ETAR receptor, integrin-linked kinase, Akt, and hypoxia-inducible factor-1α signaling pathways. 2) Knockdown of ET-1 decreases VEGF expression and inhibits angiogenesis both in vitro and in vivo. ET-1 promotes migration and tube formation of endothelial progenitor cells through increased VEGF. 3) ET-1 activates HIF-1α, which binds to the VEGF promoter region to increase VEGF expression. This ET-1/ETAR/ILK/Akt/

1. Endothelin 1 (EDN1)
Arunkumar Rengaraj
Postdoctoral Associate
Department of surgery
Miller school of Medicine
University of Miami

2. • Endothelin is an important vasodilator or Vasoconstrictive agent.
• There are three kinds of endothelin, EDN1, EDN2 and EDN3
• EDN1 is an most abundant, primarily secreted by the endothelial cells which is 22 amino acid peptide
• It is also secreted by vascular smooth muscle cells (VSMC), macrophages, and the renal medulla (1).
• Endothelin production is stimulated by physical factors, including shear stress and vascular stretch .
• Molecules such as angiotensin II (AII), antidiuretic hormone (ADH), thrombin, cytokines, reactive oxygen species.
• Endothelin gene encoded in chromosome 6, 1 and 20.
• Endothelin secreted as inactive form as Pro-Pro endothelin, Activation follows
Pro-Pro
endothelin
(212 aa)
Furin-like
proteases
Pro-
endothelin
endothelin converting
enzymes (ECEs)
Endothelin
Introduction

3. • The endothelin and receptor interaction occur by autocrine and paracrine signaling.
• Endothelin interact with the EDNRA and EDNRB receptors and activates via G-proteins or β-arrestin dependent
pathway.
• In most of the human cancers, endothelin activates MAPK, NF-kB, β-catenin, PI3K/AKT, and Rho GTPase pathways,
regulating the expression of gene sessential for cell survival, proliferation, drug resistance, angiogenesis, osteogenesis,
immune modulation, invasion and metastasis (2).
• ET-1 release is inhibited by prostacyclin and atrial natriuretic peptide as well as by nitric oxide.
https://www. wikipathways.org/index.php/Pathway:WP4857.
Introduction

4. ORIGINAL ARTICLE
Endothelin-1 promotes vascular endothelial growth
factor-dependent angiogenesis in human chondrosarcoma cells
M-H Wu1, C-Y Huang1,2,3, J-A Lin1, S-W Wang4, C-Y Peng2,5, H-C
Cheng6 and C-H Tang1,7,8
Oncogene (2014) 33, 1725–1735
& 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14
www.nature.com/onc
• Chondrosarcoma is the second most common sarcoma in bone malignancy
• Endothelin-1 (ET-1) has been implicated in tumor angiogenesis and metastasis.
• The relationship of ET-1 with vascular endothelial growth factor (VEGF) expression and angiogenesis in human mostly unknown.
• Here, we found that the expression of ET-1 and VEGF were correlated with tumor stage and were significantly higher than that in the
normal cartilage.
• Exogenous ET-1 with chondrosarcoma cells promoted VEGF expression and subsequently increased migration and tube formation in
endothelial progenitor cells.
• ET-1 increased VEGF expression and angiogenesis through ETAR, integrin-linked kinase (ILK), Akt and hypoxia-inducible factor-1a (HIF-1a)
signaling cascades.
• Knockdown of ET-1 decreased VEGF expression and also abolished chondrosarcoma conditional medium-mediated angiogenesis in vitro as
well as angiogenesis effects in the chick chorioallantois membrane and Matrigel plug nude mice model in vivo.
• In addition, in the xenograft tumor angiogenesis model, knockdown of ET-1 significantly reduced tumor growth and tumor-associated
angiogenesis.
• Taken together, these results indicate that ET-1 occurs through ETAR, ILK and Akt, which in turn activates HIF-1a, resulting in the activation of
VEGF expression and contributing to the angiogenesis and tumor growth of human chondrosarcoma cells.

5. Materials & Methods
Cell culture
The human chondrosarcoma cell line (JJ012)
Isolation and cultivation of circulating EPCs
CD34-positive EPCs
Immunohistochemistry
Human chondrosarcoma tissue array was purchased from Biomax.
ET-1 or VEGF were analyzed.
The sum of the intensity and percentage score was used as the final staining scores (0–5).
Quantitative real-time PCR
Western blot analysis
rabbit anti-human antibodies against
ILK, GSK3b, and p-GSK3b, Akt, p-Akt, HIF-1a, PCNA or VEGF (1:1000
ILK kinase assay
Briefly, ILK was immunoprecipitated with ILK antibody overnight at 4 °C from 250 µg of lysate. After immunoprecipitation, beads
were resuspended in 30 µl of kinase buffer containing 1 µg of recombinant substrate (GSK3b fusion protein) and
200 µM cold ATP, and the reaction was carried out for 30 min at 30 °C. The phosphorylated substrate was visualized by western
blot with phospho- GSK3β antibody. Total GSK3β was detected with the appropriate antibody.

6. Materials & Methods
Chromatin immunoprecipitation assay
DNA immunoprecipitated by anti-HIF-1a antibody.
DNA was then extracted with phenol–chloroform
Purified DNA pellet was subjected to PCR
Primers 50-CCTTTGGGTTTTGCCAGA-30 and 50-CCAAGTTTGTGGAGCTGA-
30 were utilized to amplify across the VEGF promoter region
Transfection and reporter assay
Human chondrosarcoma cells were transfected with reporter plasmid using Lipofectamine 2000.
At 24 h after transfection, the cells were pre-treated with inhibitors for 30 min, and then ET-1 or vehicle was added for 24 h.
Cell extracts were then prepared, and luciferase and β-galactosidase activities were measured
Preparation of CM
Tube formation assay
CAM assay
In vivo tumor xenograft study
Matrigel plug assay

7. Result
Figure 1. The correlation of ET-1, VEGF and tumor grades in human chondrosarcoma tissues. IHC of ET-1 (a) and
VEGF (b) expressions innormal cartilage and chondrosarcoma tissue. The correlation and quantitative data are
shown in (c).
The expression of ET1 and
VEGF in chondrosarcoma
patient was significantly
high.
ET1 expression strongly
correlate VEGF expression
and tumor stage.

8. Result
Figure 2. ET-1 promotes VEGF expression and subsequently increases
angiogenesis in human osteosarcoma cells. (a–c) JJ012 cells were incubated
with ET-1 (30–300 ng/ml) for 24 h, and VEGF expression was examined by
qPCR, ELISA, and western blotting. (d and e) JJ012 cells
were pre-treated for 30 min with VEGF antibody (5 mg/ml), followed by
stimulation with ET-1 (100 ng/ml) or incubated with ET-1 (10–100 ng/ ml) for
24 h. The medium was collected as CM and then applied to EPCs for 24 h. The
cell migration and capillary-like structure formation in EPCs was examined by
Transwell and tube formation assay. Results are expressed as mean±s.e.
*Po0.05 compared with control; #Po0.05 compared with ET-1-treated group
ET1 increased VEGF mRNA expression (2a)
ET1 induced VEGF secretion (2b)
ET1 treated chondrosarcoma cells cells promoted
migration and tube formation.

9. Result
Figure 3. ET-1 increases VEGF expression and angiogenesis via ERAR receptor. (a
and b) JJ012 cells were pre-treated with the BQ123 (5 mM) or BQ788 (10 mM)
for 30 min, followed by stimulation with ET-1 (100 nM) for 24 h, and VEGF
expression was examined by qPCR and ELISA. (c and d) JJ012 cells were pre-
treated with BQ123 (5 mM) or BQ788 (10 mM) for 30 min, followed by
stimulation with ET-1 (100 nM) for 24 h. The medium was collected as CM and
then applied to EPCs for 24 h. The cell migration and capillary-like structure
formation in EPCs was examined by Transwell and tube formation assay. Results
are expressed as mean±s.e. *Po0.05 compared with control; #Po0.05 compared
with ET-1- treated group.
ETAR inhibitor-BQ123; ETBR inhibitor-BQ788
BQ123 reduced VEGF mRNA, VEGF expression,
migration and tube formation.

10. Result
Figure 4. ILK/Akt pathway is involved ET-1-induced VEGF expression and
angiogenesis in human chondrosarcoma cells. (a and b) JJ012 cells were pre-
treated with the KP392 (10 mM) or Akt inhibitor (10 mM) for 30 min, followed
by stimulation with ET-1 (100 nM) for 24 h, and VEGF expression was examined
by qPCR and ELISA. (c and d) JJ012 cells were pre-treated with the KP392 (10
mM) or Akt inhibitor (10 mM) for 30 min, followed by stimulation with ET-1 (100
nM) for 24 h. The medium was collected as CM and then applied to EPCs for 24
h. The cell migration and capillary-like structure formation in EPCs was examined
by Transwell and tube formation assay. (e) JJ012 cells were incubated with ET-1
(100 ng/ml) for the indicated times, and ILK activation and Akt phosphorylation
were determined by ILK kinase assay and western blotting. Results are expressed
as mean±s.e. *Po0.05 compared with control; #Po0.05 compared with ET-1-
treated group.
ILK/Akt signaling pathway
ILK inhibitor - KP392
AKT inhibitor - (1L-6-hydroxymethylchiro-
inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate))

11. Result
Figure 5. HIF-1a transactivation is involved in ET-1-induced VEGF expression and
angiogenesis. (a and b) JJ012 cells were pre-treated with the HIF-1ainhibitor (10 mM)
for 30 min, followed by stimulation with ET-1 (100 nM) for 24 h, and VEGF expression
was examined by qPCR and ELISA. (c and d) JJ012 cells were pre-treated with the HIF-
1ainhibitor (10 mM) for 30 min, followed by stimulation with ET-1 (100 nM) for 24 h.
The medium was collected as CM and then applied to EPCs for 24 h. The cell migration
and capillary-like structure formation in EPCs was examined by Transwell and tube
formation assay. (e) JJ012 cells were incubated with ET-1 (100 ng/ml) for the indicated
times and HIF-1aaccumulation in the nucleus was determined by western blotting. (f )
JJ012 cells were incubated with ET-1 (30–300 ng/ml) for 24 h, and HIF-1a mRNA
expression was examined by qPCR. Results are expressed as mean±s.e. *Po0.05
compared with control; #Po0.05 compared with ET-1-treated group.
ET1 – HIF-1α -VEGF
HIF-1α inhibitor is utilized

12. Result
Figure 6. ETAR/ILK/Akt pathway is involved in ET-1-mediated HIF-1a activation.
(a) JJ012 cells were pre-treated with the BQ123, BQ788, KP392 and Akt
inhibitor for 30 min, followed by stimulation with ET-1 (100 nM) for 24 h, and
HIF-1a mRNA expression was examined by qPCR. (b) JJ012 cells were pre-
treated with KP392 and Akt inhibitor for 30 min, followed by stimulation with
ET-1 for 2 h, and then chromatin immunoprecipitation assay was then
performed. Chromatin was immunoprecipitated with anti-HIF-1a. The
percentage of precipitated
chromatin was assayed to verify equal loading (input). (c–e) JJ012 cells were
incubated with ET-1 (10–100 ng/ml) for 24 h or pre-treated with BQ123, BQ788,
KP392 and Akt inhibitor for 30 min or co-transfected with ILK siRNA and Akt
mutant for 24 h before exposure to ET-1. The HREdriven luciferase activity was
measured, and the results were normalized to the b-galactosidase activity and
expressed as mean±s. e. for three independent experiments performed in
triplicate. Results are expressed as mean±s.e. *Po0.05 compared with control;
#Po0.05 compared with ET-1-treated group

13. Result
Figure 7. Knockdown of ET-1 reduces angiogenetic effects in vitro and in
vivo. (a) The protein expression of ET-1 and VEGF in JJ012/control shRNA
and JJ012/ET-1-shRNA cells was examined by western blotting. (b and c)
EPCs were incubated with CM from JJ012/control-shRNA or JJ012/ET-1-
shRNA for 24 h, and tube formation or cell migration was photographed
under the microscope or examined by Transwell assay.
(d) Chick embryos were incubated with PBS, JJ012/control-shRNA CM or
JJ012/ET-1-shRNA CM for 4 days, and then resected, fixed and
photographed with a stereomicroscope. (e–g) Mice were injected
subcutaneously with Matrigel mixed with PBS, JJ012/control-shRNA CM or
JJ012/ET-1-shRNA CM for 7 days. The plugs were excised from mice and
photographed, stained with CD31 and quantified the hemoglobin content
(n¼10 in each group). Results are expressed as mean±s.e. *Po0.05
compared with control; #Po0.05 compared with ET-1-treated group.

14. Result
Figure 8. Knockdown of ET-1 decreases tumor-associated
angiogenesis in mice. (a–d) JJ012/control-shRNA or JJ012/ET-1-
shRNA cells were mixed with Matrigel and injected into flank
sites of mice for 10 days, and then resected. The tumors were
photographed with a microscope, measured weight and volume,
and quantified the hemoglobin levels. The correlation between
tumor volume and hemoglobin levels is shown in (e) (n¼10 in
each group). Results are expressed as mean±s.e. *Po0.05
compared with control.

15. Conclusion
• ET -1 increased VEGF expression and subsequently promoted angiogenesis in human chondrosarcoma cells.
• ET-1, acting through the ETAR, is associated with the progression of tumor angiogenesis.
• Stimulation of chondrosarcoma cells with ET -1 increased the activity of ILK and ET -1 mediated VEGF expression and tube
formation. Akt is the most common downstream molecule in the ILK -regulated cellular functional pathway.
• ET-1 increased VEGF production and angiogenesis by activating the ETAR, ILK, Akt, and HIF- 1α signaling pathways.
• Suppression of ET -1 expression reduced angiogenesis (CD31 on tissue) and tumor burden (weight and volume).

16. Reference
1. Titus A, Marappa-Ganeshan R. Physiology, Endothelin. [Updated 2022 May 8].
2. Dagamajalu S, A network map of endothelin mediated signaling pathway. J Cell Commun Signal.
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