Journal: Research Square

Article Title: Vactosertib, a novel TGF-β1 type I receptor kinase inhibitor, improves T-cell fitness: a single-arm, phase 1b trial in relapsed/refractory multiple myeloma

doi: 10.21203/rs.3.rs-3112163/v1

Figure Lengend Snippet: A. Vactosertib and pomalidomide dosing schedule and vactosertib dose levels for the phase 1b dose escalation cohort. Also shown is the schedule for clinical and laboratory testing. B. Shown is individual trajectories and outcomes over duration in the 19 patients that received full-dose treatment. C. Shown is the maximal change in M-spike value for the same 19 patients. Two patients (#14 and #16) were excluded from response and outcome analysis because of withdrawn consent or grade 4 toxicity.

Article Snippet: Stock solutions of vactosertib (MedChemExpress, Monmouth Junction, NJ) and internal standard (IS, valsartan, MedChemExpress) were prepared at a concentration of 10 μg/mL in 0.1% formic acid.

Techniques:

Journal: Research Square

Article Title: Vactosertib, a novel TGF-β1 type I receptor kinase inhibitor, improves T-cell fitness: a single-arm, phase 1b trial in relapsed/refractory multiple myeloma

doi: 10.21203/rs.3.rs-3112163/v1

Figure Lengend Snippet: Best overall response to therapy.

Article Snippet: Stock solutions of vactosertib (MedChemExpress, Monmouth Junction, NJ) and internal standard (IS, valsartan, MedChemExpress) were prepared at a concentration of 10 μg/mL in 0.1% formic acid.

Techniques:

Journal: Research Square

Article Title: Vactosertib, a novel TGF-β1 type I receptor kinase inhibitor, improves T-cell fitness: a single-arm, phase 1b trial in relapsed/refractory multiple myeloma

doi: 10.21203/rs.3.rs-3112163/v1

Figure Lengend Snippet: A. Effect of vactosertib, pomalidomide, and lenalidomide on MMCLs. B. Effect of vactosertib, pomalidomide, and lenalidomide on CD138 + cells isolated from MM patient BM samples. CD138 + cells were isolated using the positive selection method. Patient CD138 + cells or MMCLs (5,000 cells/well) were incubated in 96-well plates with each drug at the indicated concentration for 72 h. The effect of drugs on cell viability was determined using the XTT assay . Bioassays were performed in triplicate. Error bars represent the SE of the mean.

Article Snippet: Stock solutions of vactosertib (MedChemExpress, Monmouth Junction, NJ) and internal standard (IS, valsartan, MedChemExpress) were prepared at a concentration of 10 μg/mL in 0.1% formic acid.

Techniques: Isolation, Selection, Incubation, Concentration Assay, XTT Assay

Journal: Research Square

Article Title: Vactosertib, a novel TGF-β1 type I receptor kinase inhibitor, improves T-cell fitness: a single-arm, phase 1b trial in relapsed/refractory multiple myeloma

doi: 10.21203/rs.3.rs-3112163/v1

Figure Lengend Snippet: A. Synergistic effect of vactosertib combined with pomalidomide on drug resistant MM cells. MM patient CD138 + cells were incubated with vactosertib, pomalidomide or both for 72 h. B. Effect of vactosertib on MM cells resistant to proteasome inhibitors. RPMI8226 cells resistant to bortezomib, carfilzomib and ixazomib were generated as described . Cells were incubated alone, with each proteasome inhibitor, vactosertib or each proteasome inhibitor combined with vactosertib. C. Effect of vactosertib on MM patient CD138 + cells. Cells were incubated alone or with proteasome inhibitors, vactosertib or both. Viability was determined using the XTT assay and performed in triplicate. Error bars represent the SE of the mean.

Article Snippet: Stock solutions of vactosertib (MedChemExpress, Monmouth Junction, NJ) and internal standard (IS, valsartan, MedChemExpress) were prepared at a concentration of 10 μg/mL in 0.1% formic acid.

Techniques: Incubation, Generated, XTT Assay

Journal: Research Square

Article Title: Vactosertib, a novel TGF-β1 type I receptor kinase inhibitor, improves T-cell fitness: a single-arm, phase 1b trial in relapsed/refractory multiple myeloma

doi: 10.21203/rs.3.rs-3112163/v1

Figure Lengend Snippet: A. Effect of vactosertib and pomalidomide on cytokines. At indicated times during treatment, latent TGF-β1, free (active) TGF-β1 and IL-6 levels were measured in BM samples from N ≥ 3 patients. Error bars represent the SD of the mean. B. Effect on vactosertib and pomalidomide on the relative percentage of tumor, immune and non-immune cell types. The mononuclear cell fraction was isolated from patient BM samples and stained using antibodies specific to B cells, CD4 + and CD8 + T cells, NK cells, leukocytes, monocytes, and macrophages in patient BM samples. Cell types were quantitated by immunostaining followed by flow cytometry. Cell types were quantitated from N ≥3 MM patients. Error bars represent the SD of the mean. C. Effect on vactosertib and pomalidomide on the relative surface expression of immunosuppressive markers on patient CD8 + T-cells. Relative levels of PD-1, TIM-3, 2B4, BTLA, and CTLA4 were measured at indicated times. Asterisks indicate statistical significance ( p ≤ 0.05).

Article Snippet: Stock solutions of vactosertib (MedChemExpress, Monmouth Junction, NJ) and internal standard (IS, valsartan, MedChemExpress) were prepared at a concentration of 10 μg/mL in 0.1% formic acid.

Techniques: Isolation, Staining, Immunostaining, Flow Cytometry, Expressing

Journal: Research Square

Article Title: Vactosertib, a novel TGF-β1 type I receptor kinase inhibitor, improves T-cell fitness: a single-arm, phase 1b trial in relapsed/refractory multiple myeloma

doi: 10.21203/rs.3.rs-3112163/v1

Figure Lengend Snippet: A. Shown is the relative MFI value for PD-1, TIM-3, BTLA, and LAG3 expression on CD8 + T-cells isolated from peripheral blood of healthy donors. T-cells were cultured in the presence of TGF-β1 at the indicated concentrations. Error bars represent the standard error of the mean. CTLA-4 was not detectable by immunostaining on healthy T-cells. Error bars represent the SD of the mean. B. Shown is relative MFI values after staining for PD-1, TIM-3, BTLA, and LAG3 on MM patient CD8 + T-cells. Error bars represent the SD of the mean. C. Effect of vactosertib on MM patient CD8 + T-cell viability. MM patient CD8 + T-cells were treated vactosertib and/or pomalidomide for 48 h. XTT assay solution was added to the plate and incubated in dark for 3 h. The relative cell viability was quantified by measuring the absorbance at 450 nm on SpectraMax i3x multi-mode microplate reader. D. Effect of vactosertib on TGFβ levels in patient CD8 + T-cell cultures. CD8 + T-cells were cultured with vactosertib, pomalidomide and combinations for 48 h. Cells were then centrifuged for 5 min at 3,000 × rpm. Supernatants were removed and TGF-β1 levels were determined using the human TGF-β1 duoSet ELISA kit. Error bars represent the SE of the mean. E. Effect of vactosertib on TGF-β1 levels in MMCL supernatants. Cells were cultured with vactosertib, pomalidomide and combinations as shown for 48 h. Cells were centrifuged for 5 min (3,000 × rpm), supernatants removed and TGF-β1 levels determined using the human TGF-β1 duoSet ELISA kit. F. Effect of vactosertib on TGF-β1 levels in patient CD138 + supernatants. Cells were cultured with vactosertib, pomalidomide and combinations for 48 h as shown, centrifuged for 5 min at 3,000 × rpm and supernatants removed. TGF-β1 levels were determined using the human TGF-β1 duoSet ELISA kit. G. Effect of vactosertib on PD-L1 and PD-L2 expression on MMCLs. MMCLs were treated with vactosertib and/or pomalidomide at indicated concentrations for 72 h. Cells were then stained with PD-L1 and PD-L2-specific antibodies simultaneously for 20 min and analyzed by flow cytometry. H . Effect of vactosertib on PD-L1 and PD-L2 expression on MM patient CD138 + cells. Patient CD138 + cells were treated with vactosertib and/or pomalidomide at various concentrations for 48 h. Cells were then stained with PD-L1 and PD-L2 specific antibodies simultaneously for 20 min and analyzed by flow cytometry. I. Effect of vactosertib on autologous T-cell cytotoxic activity. Shown is the cytotoxic effect of autologous CD8 + T-cells from patient peripheral blood on patient CD138 + cells following 24 h co-culture. CD138 + cells (20,000/well) were treated with drugs as indicated for 8 h and then co-cultured CD8 + T-cells (50,000/well) for 18 h. Cells were then stained with a CD138 + -specific antibody, followed by propidium iodide and annexin-V for 15 min in the dark. Apoptosis was quantified in cells positively gated for CD138 + , annexin-V and propidium iodide by flow cytometry using FlowJo_10.8.1 software. Black bars represent the effect of T-cells alone. Asterisks indicate statistical significance ( p ≤ 0.05). Error bars represent the SD of the mean.

Article Snippet: Stock solutions of vactosertib (MedChemExpress, Monmouth Junction, NJ) and internal standard (IS, valsartan, MedChemExpress) were prepared at a concentration of 10 μg/mL in 0.1% formic acid.

Techniques: Expressing, Isolation, Cell Culture, Immunostaining, Staining, XTT Assay, Incubation, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Activity Assay, Co-Culture Assay, Software

Journal: Circulation

Article Title: Aldosterone Enhances Ischemia-Induced Neovascularization Through Angiotensin II–Dependent Pathway

doi: 10.1161/01.cir.0000127112.36796.9b

Figure Lengend Snippet: Figure 1. Representative photomicro- graphs and quantitative evaluation of microangiography (A), capillary density (B; capillary appears in white, and arrows indicate representative examples of fibronectin-positive capillaries), and foot perfusion (C). D, Representative pho- tomicrographs of Western blot and quantitative evaluation of VEGF protein content. Values are meanSEM; n7. *P0.05 vs control mice; #P0.05 vs aldosterone-treated mice; †P0.05 vs nonischemic control. C indicates untreated animals; A, aldosterone-treated mice; AS, aldosterone and spironolac- tone–treated mice; AV, aldosterone and valsartan–treated mice; Aa-VEGF, aldo- sterone and neutralizing VEGF–treated mice; Mb, membrane stained with pon- ceau red; N. Isch., nonischemic; Isch., ischemic; Cont, control; and Aldo, aldosterone.

Article Snippet: C57Bl/6 mice (aged 10 weeks; Iffa Creddo, Lyon, France) were anesthetized by isoflurane inhalation, and unilateral hindlimb ischemia was induced by ligature (tied for complete occlusion) on the right femoral artery, as previously described).4,6 Mice were then randomly assigned to one of the following groups (n 7): (1) control group: vehicle (1% ethanol in drinking water); (2) mice receiving the mineralocorticoid receptor blocker spironolactone (spironolactone dissolved in ethanol at 25 mg/mL and added to drinking water; mice then received 20 mg/kg per day; Sigma); (3) mice treated with the AT1 receptor blocker valsartan (drinking water, 20 mg/kg per day; Sigma); (4) aldosterone (4.5 g/d by osmotic minipumps implanted subcutaneously in the back of the mice; model 2004, Alza Corp); (5) aldosterone plus spironolactone; (6) aldosterone plus valsartan; and (7) aldosterone plus a-VEGF neutralizing antibody (10 g IP twice per week; R&D Systems).

Techniques: Western Blot, Control, Membrane, Staining

Journal: Journal of Inflammation Research

Article Title: Valsartan Mitigates the Progression of Methotrexate-Induced Acute Kidney Injury in Rats via the Attenuation of Renal Inflammation and Oxidative Stress

doi: 10.2147/JIR.S456610

Figure Lengend Snippet: Semiquantitative Histopathological Scoring Analysis of Changes in the Kidneys of Rats

Article Snippet: Methotrexate and Valsartan were purchased from TargetMol, Massachusetts, United States (MolPort-003-665-521 and MolPort-003-666-608 respectively) and dissolved in DMSO (dimethyl sulfoxide; Millipore, Molsheim, France).

Techniques: Control

Journal: Free radical biology & medicine

Article Title: Angiotensin II type 1 receptor blockade suppresses light-induced neural damage in the mouse retina.

doi: 10.1016/j.freeradbiomed.2014.03.020

Figure Lengend Snippet: Fig. 1. Suppression of light-induced visual function impairment by ARBs. Analysis of full-field ERG after light exposure. (A) Representative wave forms of the ERG from an individual mouse treated with one of the ARBs, valsartan, in each dosage group in response to one flash. (B, C) Amplitudes of the a-wave and b-wave were decreased 6 days after light exposure, and these changes were suppressed by treatment with ARBs in a dose-dependent manner. (D, E) No differences were observed in the a-wave or b-wave implicit times. ERG, electroretinogram; ARBs, angiotensin II type 1 receptor blockers; C, control; LE, light exposure; val, valsartan; los, losartan; can, candesartan. n ¼ 6 in each group. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. **P o 0.01, *P o 0.05.

Article Snippet: Valsartan (LKT Laboratories, St. Paul, MN, USA) and candesartan (Sigma–Aldrich, St. Louis, MO, USA) were solubilized in dimethyl sulfoxide (Sigma–Aldrich), followed by dilution with phosphatebuffered saline (PBS).

Techniques: Control

Journal: Free radical biology & medicine

Article Title: Angiotensin II type 1 receptor blockade suppresses light-induced neural damage in the mouse retina.

doi: 10.1016/j.freeradbiomed.2014.03.020

Figure Lengend Snippet: Fig. 2. Suppression of light-induced histological changes in the retina by valsartan. (A) H-E staining of retinal sections 6 days after light exposure. (B) The ONL thickness and (C) the OS length in the retina of light-exposed mice were reduced compared with those of untreated control mice. This reduction was significantly attenuated by valsartan administration (5 mg/kg). (D, E) TUNEL assay performed 2 days after light exposure. TUNEL-positive cells (red) appeared in the ONL after light exposure. These apoptotic cells were significantly reduced by valsartan administration (5 mg/kg). Hoechst staining of the control was shown as a guide for the retinal layers. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment; ■, control mice with no light exposure; ●, light-exposed mice treated with vehicle; ○, light-exposed mice treated with valsartan. n ¼ 6 in each group. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. **P o 0.01, *P o 0.05.

Article Snippet: Valsartan (LKT Laboratories, St. Paul, MN, USA) and candesartan (Sigma–Aldrich, St. Louis, MO, USA) were solubilized in dimethyl sulfoxide (Sigma–Aldrich), followed by dilution with phosphatebuffered saline (PBS).

Techniques: Staining, Control, TUNEL Assay

Journal: Free radical biology & medicine

Article Title: Angiotensin II type 1 receptor blockade suppresses light-induced neural damage in the mouse retina.

doi: 10.1016/j.freeradbiomed.2014.03.020

Figure Lengend Snippet: Fig. 3. Inhibition of light-induced ROS accumulation by treatment with valsartan. Detection of ROS by DHE staining 1 h after light exposure. (A, B) The fluorescence intensity of DHE in the ONL measured by ImageJ was increased after light exposure. The light-induced increase in ROS levels was prevented by treatment with valsartan (5 mg/kg). ONL, outer nuclear layer. n ¼ 6 in each group. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. **P o 0.01, *P o 0.05.

Article Snippet: Valsartan (LKT Laboratories, St. Paul, MN, USA) and candesartan (Sigma–Aldrich, St. Louis, MO, USA) were solubilized in dimethyl sulfoxide (Sigma–Aldrich), followed by dilution with phosphatebuffered saline (PBS).

Techniques: Inhibition, Staining

Journal: Free radical biology & medicine

Article Title: Angiotensin II type 1 receptor blockade suppresses light-induced neural damage in the mouse retina.

doi: 10.1016/j.freeradbiomed.2014.03.020

Figure Lengend Snippet: Fig. 5. Attenuation of light-induced molecular changes by treatment with either valsartan or NAC. (A, B) The mRNA level of c-fos measured by quantitative real-time RT-PCR, 1 h after light exposure, was significantly attenuated in the retinas of light-exposed mice treated with either (A) valsartan or (B) NAC, compared with vehicle-treated mice. (C, D) The increase in the fasl mRNA level 6 h after light exposure was significantly attenuated in the retinas of light-exposed mice treated with either (C) valsartan or (D) NAC, compared with vehicle-treated mice. Valsartan was administered at 5 mg/kg and NAC was at 250 or 500 mg/kg. The NAC-induced suppression was dose-dependent (B, D). NAC, N-acetyl-L-cysteine. n ¼ 6. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. **P o 0.01, *P o 0.05.

Article Snippet: Valsartan (LKT Laboratories, St. Paul, MN, USA) and candesartan (Sigma–Aldrich, St. Louis, MO, USA) were solubilized in dimethyl sulfoxide (Sigma–Aldrich), followed by dilution with phosphatebuffered saline (PBS).

Techniques: Quantitative RT-PCR