Objective:To investigate whether paclitaxel (PTX) can induce immunogenic cell death (ICD) in vascular smooth muscle cells (VSMCs), and explore the new molecular mechanism of PTX-coated balloon angioplasty in intracranial atherosclerotic stenosis.Methods:(1) Cell culture and identification: VSMCs were induced into synthetic vascular smooth muscle cells (sVSMCs); the mRNA and protein expressions of smooth muscle protein 22-α (SM22-α) and α-smooth muscle actin (α-SMA) in VSMCsS and sVSMCs were detected by real-time fluorescent quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blotting, respectively. Human acute monocytic leukemia cell line THP-1 was induced into dendritic cells (DCs); the CD86 and CD83 expressions in THP-1 and DCs were detected by flow cytometry. (2) Cell viability detection: cell counting kit-8 (CCK-8) assay was used to detect the cell viability of sVSMCs after 0, 0.01, 0.05, 0.5, 5, 10, 50, and 100 μmol/L PTX or under 0, 50, 100, 200, 400, and 600 mmHg (1 mmHg=0.133 kPa) pressures. (3) ICD marker detection: sVSMCs were collected and divided into blank-control group, dimethyl sulfoxide (DMSO) group and PTX group (cultured with 3.2 μmol/L PTX) at normal state and pressure procedure (188 mmHg), respectively; calreticulin (CRT) expression was detected by immunofluorescent staining; adenosine triphosphate (ATP) expression was detected by luciferase assay, and high mobility group protein B1 (HMGB1) expression was detected by enzyme-linked immunosorbent assay (ELISA). (4) ICD-related immune activation assay detection: sVSMCs and DCs were collected and divided into DCs group, PTX+DCs group (cultured with 3.2 μmol/L PTX), DCs+sVSMCs group, and PTX+DCs+sVSMCs group (cultured with 3.2 μmol/L PTX); CD86 and CD83 expressions were detected by flow cytometry; interleukin (IL)-2, IL-10 and interferon-γ (IFN-γ) levels were detected by ELISA. The sVSMCs, DCs and CD8 +T cells were collected and divided into sVSMCs group, sVSMCs+DCs group, sVSMCs+CD8 +T cell group, sVSMCs+DCs+CD8 +T cell group, PTX+sVSMCs group (cultured with 3.2 μmol/L PTX), and PTX+sVSMCs+DCs+CD8 +T cell group (cultured with 3.2 μmol/L PTX); proliferation of these cells was detected by cell clone formation assay. Results:(1) The SM22-α and α-SMA mRNA and protein expressions in the sVSMCs group were significantly lower than those in the VSMCs group ( P<0.05); rate of double-positive CD83 and CD86 in the DCs group was significantly higher than that in the THP-1 group ( P<0.05). (2) The sVSMCs viability decreased in a concentration-dependent manner after PTX treatment at concentrations of 0, 0.01, 0.05, 0.5, 5, 10, 50, and 100 μmol/L, respectively, with significant differences ( P<0.05); half maximal inhibitory concentration (IC 50) of PTX on sVSMCs was 3.2 μmol/L; no significant difference in sVSMCs viability after 3.2 μmol/L PTX treatment was noted under 0, 50, 100, 200, 400, and 600 mmHg pressures ( P>0.05). (3) Under normal state and pressure procedure, CRT fluorescent intensity of sVSMCs in the PTX group (42.00±3.50, 24.19±2.41) was significantly higher than that in the blank-control group (8.60±1.8, 8.42±1.7) and DMSO group (10.23±1.47, 9.71±1.01), ATP luminescence intensity (17 399.33±2 035.58, 17 445.67±2 449.34) was significantly higher than that in the blank-control group (9 021.33±726.84, 10 271.33±2 194.22) and DMSO group (11 977.33±960.91, 11 683.33±419.50), and HMGB1 concentration ([3 258.31±502.08] pg/mL, [3 265.27±246.06] pg/mL) was significantly higher than that in the blank-control group ([1 156.48±184.96] pg/mL, [1 205.20±196.36] pg/mL) and DMSO group ([1 309.59±75.03] pg/mL, [1 265.51±14.52] pg/mL, P<0.05). (4) The PTX+DCs+sVSMCs group had significantly higher CD83, CD86, IFN-γ and IL-2 expressions and lower IL-10 expression than the DCs group, PTX+DCs group, and DCs+sVSMCs group ( P<0.05); the PTX+sVSMCs group and PTX+sVSMCs+DCs+CD8 +T cell group had significantly lower clone formation rate compared with the sVSMCs group, sVSMCs+DCs group, sVSMCs+CD8 +T cell group, and sVSMCs+DCs+CD8 +T cell group ( P<0.05). Conclusion:PTX can promote ICD in VSMCs by promoting DCs activation and enhancing CD8 +T cell toxicity.

Objective:To investigate whether paclitaxel (PTX) can induce immunogenic cell death (ICD) in vascular smooth muscle cells (VSMCs), and explore the new molecular mechanism of PTX-coated balloon angioplasty in intracranial atherosclerotic stenosis.Methods:(1) Cell culture and identification: VSMCs were induced into synthetic vascular smooth muscle cells (sVSMCs); the mRNA and protein expressions of smooth muscle protein 22-α (SM22-α) and α-smooth muscle actin (α-SMA) in VSMCsS and sVSMCs were detected by real-time fluorescent quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blotting, respectively. Human acute monocytic leukemia cell line THP-1 was induced into dendritic cells (DCs); the CD86 and CD83 expressions in THP-1 and DCs were detected by flow cytometry. (2) Cell viability detection: cell counting kit-8 (CCK-8) assay was used to detect the cell viability of sVSMCs after 0, 0.01, 0.05, 0.5, 5, 10, 50, and 100 μmol/L PTX or under 0, 50, 100, 200, 400, and 600 mmHg (1 mmHg=0.133 kPa) pressures. (3) ICD marker detection: sVSMCs were collected and divided into blank-control group, dimethyl sulfoxide (DMSO) group and PTX group (cultured with 3.2 μmol/L PTX) at normal state and pressure procedure (188 mmHg), respectively; calreticulin (CRT) expression was detected by immunofluorescent staining; adenosine triphosphate (ATP) expression was detected by luciferase assay, and high mobility group protein B1 (HMGB1) expression was detected by enzyme-linked immunosorbent assay (ELISA). (4) ICD-related immune activation assay detection: sVSMCs and DCs were collected and divided into DCs group, PTX+DCs group (cultured with 3.2 μmol/L PTX), DCs+sVSMCs group, and PTX+DCs+sVSMCs group (cultured with 3.2 μmol/L PTX); CD86 and CD83 expressions were detected by flow cytometry; interleukin (IL)-2, IL-10 and interferon-γ (IFN-γ) levels were detected by ELISA. The sVSMCs, DCs and CD8 +T cells were collected and divided into sVSMCs group, sVSMCs+DCs group, sVSMCs+CD8 +T cell group, sVSMCs+DCs+CD8 +T cell group, PTX+sVSMCs group (cultured with 3.2 μmol/L PTX), and PTX+sVSMCs+DCs+CD8 +T cell group (cultured with 3.2 μmol/L PTX); proliferation of these cells was detected by cell clone formation assay. Results:(1) The SM22-α and α-SMA mRNA and protein expressions in the sVSMCs group were significantly lower than those in the VSMCs group ( P<0.05); rate of double-positive CD83 and CD86 in the DCs group was significantly higher than that in the THP-1 group ( P<0.05). (2) The sVSMCs viability decreased in a concentration-dependent manner after PTX treatment at concentrations of 0, 0.01, 0.05, 0.5, 5, 10, 50, and 100 μmol/L, respectively, with significant differences ( P<0.05); half maximal inhibitory concentration (IC 50) of PTX on sVSMCs was 3.2 μmol/L; no significant difference in sVSMCs viability after 3.2 μmol/L PTX treatment was noted under 0, 50, 100, 200, 400, and 600 mmHg pressures ( P>0.05). (3) Under normal state and pressure procedure, CRT fluorescent intensity of sVSMCs in the PTX group (42.00±3.50, 24.19±2.41) was significantly higher than that in the blank-control group (8.60±1.8, 8.42±1.7) and DMSO group (10.23±1.47, 9.71±1.01), ATP luminescence intensity (17 399.33±2 035.58, 17 445.67±2 449.34) was significantly higher than that in the blank-control group (9 021.33±726.84, 10 271.33±2 194.22) and DMSO group (11 977.33±960.91, 11 683.33±419.50), and HMGB1 concentration ([3 258.31±502.08] pg/mL, [3 265.27±246.06] pg/mL) was significantly higher than that in the blank-control group ([1 156.48±184.96] pg/mL, [1 205.20±196.36] pg/mL) and DMSO group ([1 309.59±75.03] pg/mL, [1 265.51±14.52] pg/mL, P<0.05). (4) The PTX+DCs+sVSMCs group had significantly higher CD83, CD86, IFN-γ and IL-2 expressions and lower IL-10 expression than the DCs group, PTX+DCs group, and DCs+sVSMCs group ( P<0.05); the PTX+sVSMCs group and PTX+sVSMCs+DCs+CD8 +T cell group had significantly lower clone formation rate compared with the sVSMCs group, sVSMCs+DCs group, sVSMCs+CD8 +T cell group, and sVSMCs+DCs+CD8 +T cell group ( P<0.05). Conclusion:PTX can promote ICD in VSMCs by promoting DCs activation and enhancing CD8 +T cell toxicity.

OBJECTIVES: The 5MAX ACE is a new large bore aspiration catheter available for vessel recanalization for treatment of acute ischemic stroke (AIS). We report our initial experience with its use. METHODS: A retrospective analysis of patients undergoing intra-arterial therapy for AIS using the 5MAX ACE reperfusion catheter at our institution was performed. Patient demographics, clinical characteristics and procedural data were obtained from chart review. Successful recanlization was defined as achievement of Thrombolysis in Cerebral Infarction score (TICI) 2b-3 and time to recanalization was defined as time from groin puncture to achievement of at least TICI 2b recanalization. RESULTS: The 5MAX ACE was used in 15 patients from July-October 2013. Direct aspiration was used as the primary technique in 10/15 (67%) patients. Out of these, aspiration alone was sufficient for recanalization in 3 (20%) patients. In the remaining 7 (47%) patients, additional devices were used. In 5/15 (33%) patients, combined aspiration/stentriever thrombectomy using Solitaire(TM) (3/5 patients) and Penumbra 3D Separator(TM) (2/5 patients) were used as the primary technique. Successful recanlization (TICI 2b-3) was achieved in 11/15 (73%) patients. Average time from groin puncture to successful recanalization was 46 +/- 30 minutes (range 14-98 minutes). There were no procedural complications. CONCLUSION: The 5MAX ACE is a useful recanalization tool, either by direct aspiration or combined stentriever/aspiration. It may be most advantageous with large clots in the internal carotid artery. The potential for effective and faster recanalization using this device alone or in combination may be a good topic for future study.
Carotid Artery, Internal ; Catheters* ; Cerebral Infarction ; Cerebral Revascularization ; Demography ; Endovascular Procedures ; Groin ; Humans ; Punctures ; Reperfusion* ; Retrospective Studies ; Stroke* ; Suction ; Thrombectomy