Journal: Cancers

Article Title: Transient Receptor Potential Channel Expression Signatures in Tumor-Derived Endothelial Cells: Functional Roles in Prostate Cancer Angiogenesis

doi: 10.3390/cancers11070956

Figure Lengend Snippet: Identification of the trp expression signature in PTEC. ( a ) Heatmap showing the relative mRNA expression of the trp genes in TECs of different origins. TEC expression normalized based on normal endothelial counterparts: PTEC1-3 expression is normalized against HPrMEC expression, while BTEC and RTEC expression is normalized against HMEC and GEC expression, respectively. ( b ) Differential analysis of PTEC1-3 and HPrMEC highlighted six genes (i.e., trpa1 , trpc1 , trpc4 , trpm1 , trpm7 , and trpml1 ) that are differentially deregulated (adjusted p -value < 0.1, red line), while the other genes did not show significant deregulation. ( c ) Differential analysis identified six genes that are differentially deregulated (panels a , b ), and twelve genes displayed at least 2-fold change in their expression in at least two patient lines (panel e ). Among these, four channels ( trpa1 , trpv2 , trpc3 and trpc6 ) were selected as ‘ prostate-associated ’. ( d ) Real-time qPCR analysis of mRNA expression shows that trpa1 , trpv2 , trpc3 and trpc6 are deregulated in PTEC1-3 relative to those in normal HPrMEC, and the deregulation is observed only in the prostatic tissue but not in the other normal ECs or TECs tested. Values are expressed as the mean ΔΔCT values ± SEM of at least three independent experiments. *: p -value < 0.05. ( e ) Scatter plots showing the expression of trp channels in PTEC1 (left panel), PTEC2 (central panel) and PTEC3 (right panel) relative to that in HPrMEC, as determined by real-time qPCR. Gray dots indicate genes with less than 2-fold change in PTEC relative to those in HPrMEC. Blue dots indicate genes with more than 2-fold change in at least one PTEC line relative to those in HPrMEC. Red dots showing the four ‘ prostate-associated ’ genes (i.e., trpa1 , trpv2 , trpc3 , and trpc6 ), with more than 2-fold change in at least 2 PTEC lines out of three. Correlation values for trp channels are expressed as Log (C_(T,gene x)/C_(T,18s)) in primary PTEC ( x -axis) and normal HPrMECs ( y -axis).

Article Snippet: Normal human prostatic microvascular ECs (HPrMEC) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA).

Techniques: Expressing

Journal: Cancers

Article Title: Transient Receptor Potential Channel Expression Signatures in Tumor-Derived Endothelial Cells: Functional Roles in Prostate Cancer Angiogenesis

doi: 10.3390/cancers11070956

Figure Lengend Snippet: Protein expression of ‘ prostate-associated ’ genes in PTEC and HPrMEC. ( a ) (panel a i , ×20) TRPA1 expression in normal areas: focal positive staining in epithelial cells (red arrows) and diffuse staining in ECs (blue arrows). (panel a ii , ×20) In tumor areas, TRPA1 expression is observed in all cases ( n = 10/10) in ECs (blue arrows), with negative staining in tumor cells (red arrows). (panel b i , ×40) In histologically normal areas, TRPV2 expression is not observed in ECs ( n = 0/10) (blue arrows) or epithelial cells (red arrows). (panel b ii , ×40) Positive endothelial TRPV2 expression was observed in 7 of 10 cases in the tumor areas (blue arrows), with positive staining in cancer cells in 4 cases (red arrow). TRPC3 expression: (panel c i , ×40) all normal tissues showed negative staining ( n = 10/10) in ECs (blue arrows) with focal positive staining in epithelial cells (red arrow). (panel c ii , ×10) Only one tumor ( n = 1/10) showed faint positive TRPC3 staining on ECs (blue arrow), whereas cancer cells were positive (red arrows) in all cases. (panel d i , ×20) TRPC6 expression was observed in ECs in histologically normal tissues ( n = 6/10), (panel d ii , ×40) whereas TRPC6 expression was absent in cancer cells in 7 out of 10 patient samples (red arrows). ( b ) Summary of endothelial and epithelial expression of the four ‘ prostate-associated ’ candidates in both normal and tumor areas for the ten patient samples used for immunohistochemical analysis. The patients were 62 to 70 years old, with PCa classified as ISUP group 2 in six cases and group 3 in four cases. Ca 2+ - imaging traces in response to TRPA1, TRPV2 and TRPC3 agonists respectively ( c ) 20 µM AITC), ( d ) 10 µM LPC and ( e ) 50 µM OAG in PTEC1 (black), PTEC2 (blue) and PTEC3 (red) but not in HPrMEC (green).Traces represents mean ± SEM of cells in the recorded field of one representative experiment. Lower panel: histogram showing peak amplitude of TRP agonist-mediated Ca 2+ responses (mean ± 95% CI of different cells in the field from at least 3 independent experiments). Statistical significance and ****: p -value < 0.0001 (Kruskal-Wallis test).

Article Snippet: Normal human prostatic microvascular ECs (HPrMEC) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA).

Techniques: Expressing, Staining, Negative Staining, Immunohistochemical staining, Imaging

Journal: Cancers

Article Title: Transient Receptor Potential Channel Expression Signatures in Tumor-Derived Endothelial Cells: Functional Roles in Prostate Cancer Angiogenesis

doi: 10.3390/cancers11070956

Figure Lengend Snippet: hTERT PTEC validation as cellular model for TEC studies. ( a ) Real-time qPCR analysis validating equal amount of mRNA expression if trpa1 , trpv2 , trpc3 and trpc6 in hTERT PTEC compared to PTEC1-3, as well as deregulated expression compared to HMEC and HPrMEC. Values are expressed as the mean ΔΔCT values ± SEM of three independent experiments. ( b ) Representative immunoblots blots ( n = 3) showing similar TRPA1, TRPV2 and TRPC3, and TRPC6 expression in hTERT PTEC compared to PTEC1, 2 and 3. Β-actin was used as a loading control. ( c ) Ca 2+ - imaging traces in response to TRPA1, TRPV2 and TRPC3 agonists respectively 20 µM AITC), 10 µM LPC and 50 µM OAG in HMEC (red), HPrMEC (green) and hTERT PTEC (black). Traces represents mean ± SEM of all cells in the recorded field of one representative experiment. Lower panel: histogram showing peak amplitude of TRP agonist-mediated Ca 2+ responses (median ± 95% CI of different cells in the field from at least three independent experiments). Statistical significance *: p -value < 0.05, **: p -value < 0.005 and ****: p -value < 0.0001 (Kruskal-Wallis test.).

Article Snippet: Normal human prostatic microvascular ECs (HPrMEC) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA).

Techniques: Biomarker Discovery, Expressing, Western Blot, Control, Imaging

Journal: Cancers

Article Title: Transient Receptor Potential Channel Expression Signatures in Tumor-Derived Endothelial Cells: Functional Roles in Prostate Cancer Angiogenesis

doi: 10.3390/cancers11070956

Figure Lengend Snippet: TRPA1 promotes in vitro tubulogenesis and in vivo angiogenesis. ( a ) left panels: TRPA1-overexpressing HMEC display increased in vitro tubulogenesis in Matrigel compared to control cells; right panels: TRPA1-downregulating hTERT PTEC display decreased in vitro tubulogenesis in Matrigel/collagenI substrate compared to control cells; bar graphs show the quantification of number of master segment and the number of isolated segments of control HMECs and TRPA1-overexpressing HMECs. Three independent experiments were performed 24 h after overexpression or after II oligofection pulse. ( b ) Endogenous TRPA1 is diffusely expressed in postnatal mouse retina but clearly co-localizes with CD31 staining, indicating that TRPA1 is found in ECs. ( c , upper panel): analysis of CD31 levels by qPCR in a fraction of CD31-enriched cell population isolated from the mouse retina (CD31+) compared to the depleted cell population (CD31−); ( c , lower panel): analysis of TRPA1 mRNA expression by qPCR in a fraction of CD3-enriched cell population isolated from the mouse retina (CD31+) compared to the depleted cell population (CD31−). Values are expressed relative to actin expression. Statistical significance *: p -value < 0.05, **: p -value < 0.005 and ***: p -value < 0.0005 vs. control. ( d ) Subretinal injection of AITC or HC030031 did not alter the overall development of the vascular plexus 3 days after treatment, but the front of growing vasculature became uneven after injection of the TRPA1 agonist AITC in the subretinal space. ( e , upper panel) This was illustrated by a significant increase of 26.8% in the length of sprouts (white bars): quantification of tip cell length during mouse retinal angiogenesis shows that compared to that in controls, tip cell length increased significantly 3 days post-intravitreous injection of 200 µM AITC. ( e , lower panel): Treatment with TRPA1 inhibitor (200 µM HC030031) induced a decrease in tip cell length. Cell lengths were either classified into three groups according to ranges (<70 µm, 70–150 µm, >150 µm) or considered altogether (total). Bars represent the mean ± SEM of each length range or total grouping. ( f , upper panel): Percentage of tip cells with each length range shows that treatment with AITC induces a decrease in the number of short tip cells (<70 µm) and an increase in the number of medium and long tip cells (>70 µm). ( f , lower panel): Inhibition of TRPA1 with HC030031 induces the opposite effect, increasing the number of short tip cells and decreasing the number of medium and long tip cells.

Article Snippet: Expression profiles of TECs were compared to those of several normal human ECs, namely, human umbilical vein ECs (HUVECs), human microvascular ECs from derma (HMECs), glomerular ECs from the kidney (GECs), and the commercially available human prostatic microvascular ECs (HPrMEC, ScienCell Research Laboratories, Carlsbad, CA, USA).

Techniques: In Vitro, In Vivo, Control, Isolation, Over Expression, Staining, Expressing, Injection, Inhibition

Journal:

Article Title: Differential effects of VEGFR-1 and VEGFR-2 inhibition on tumor metastases based on host organ environment

doi: 10.1158/0008-5472.CAN-10-1138

Figure Lengend Snippet: A, Western blot analysis for total and phospho-VEGFR-1 and VEGFR-2 in human lung microvascular ECs (Lung EC) and human liver sinusoidal EC (Liver EC) lysates. VEGF, neutralizing antibody to VEGFR-1 (αVEGFR-1), and neutralizing antibody to VEGFR-2 (αVEGFR-2) were added where indicated. β-actin blot serves as loading control. B, quantification of VEGFR-1 and VEGFR-2 phosphorylation relative to total VEGFR-1 and VEGFR-2 expression. C, EC proliferation (using MTT assay), migration (using modified Boyden chamber), and capillary tube formation (using Matrigel) for Lung EC and Liver EC. VEGF, αVEGFR-1, and/or αVEGFR-2 were added where indicated. All data are represented as a percentage with the addition of VEGF alone given a value of 100%. Bars represent standard deviation. P-values in text.

Article Snippet: Human liver sinusoidal ECs (Liver EC) and human lung microvascular ECs (Lung EC) were obtained from ScienCell.

Techniques: Western Blot, Control, Phospho-proteomics, Expressing, MTT Assay, Migration, Modification, Standard Deviation