Induced pluripotent stem cells can improve thrombolytic effect of low-dose rt-PA after acute carotid thrombosis in rat

Abstract

Background: Intravenous thrombolysis using recombinant tissue plasminogen activator (rt-PA) is the standard treatment for acute ischemic stroke. Standard-dose rt-PA (0.9 mg/kg) is known to achieve good recanalization but carries a high bleeding risk. Lower dose of rt-PA has less bleeding risk but carries a high re-occlusion rate. We investigate if induced pluripotent stem cells (iPSCs) can improve the thrombolytic effect of low-dose rt-PA (0.45 mg/kg).

Methods: Single irradiation with 6 mW/cm² light-emitting diode (LED) for 4 h at rat common carotid artery was used as thrombosis model according to our previous report. Endothelin-1 (ET-1), intercellular adhesion molecule-1 (ICAM-1), and interleukin 1 beta (IL-1 beta) were used as the inflammatory markers for artery endothelial injury. Angiopoietin-2 (AP-2), brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) were examined in artery wall and iPSCs culture. Animal ultrasound was used to evaluate the stenosis degree of common carotid artery before and at 2 h, 24 h, 4 days and 7 days after LED irradiation.

Results: After LED irradiation alone, there was a persistent occlusion from 2 h to 7 days. Standard-dose rt-PA alone could recanalize the occluded artery from 24 h to 7 days to stenotic degree ≤ 50%. Low-dose rt-PA or 1 × 10⁶ mouse iPSCs alone could not recanalize the occluded arteries from 2 h to 7 days. Combination use of low-dose rt-PA plus 1 × 10⁶ mouse iPSCs caused better recanalization from 24 h to 7 days. ET-1, ICAM-1 and IL-1 beta were strongly expressed after LED irradiation but reduced after iPSCs treatment. AP-2, BDNF and VEGF were rarely induced after LED irradiation but strongly expressed after iPSCs treatment. In vitro study showed iPSCs could express AP-2, BDNF and VEGF.

Conclusion: The adjuvant use of iPSCs may help improving the thrombolytic effect of low-dose rt-PA by suppressing inflammatory factors and inducing angiogenic trophic factors. Stem cells could be a potential regimen in acute thrombolytic therapy to improve recanalization and reduce complications.

Keywords: Carotid thrombosis; Endothelium; Stem cell; Thrombolysis; Tissue plasminogen activator.

Fig. 1

Schema of experimental procedure. Experimental procedure of the five treatment groups (n = 8 in each group) is demonstrated. AP-2 = angiopoietin-2; BDNF = brain-derived neurotrophic factor; ELISA = enzyme-linked immunosorbent assay; ET-1 = endothelin-1; ICAM-1 = intercellular adhesion molecule-1; ICF = immunoconfocal staining; IHC = immunohistochemistry; IL-1 beta = interleukin-1 beta; LED = light-emitting diode; miPSCs = mouse induced pluripotent stem cells; rt-PA = recombinant tissue plasminogen activator; SSEA1 = stage-specific embryonic antigen 1; US = small-animal carotid ultrasound study; VEGF = vascular endothelial growth factor. h = hour; d = day

Fig. 2

Ultrasound studies of common carotid artery (CCA) and the ratio of recanalization to ≤ 50% stenosis in the 5 treatment groups at 4 time points after 6 mW/cm² LED irradiation for 4 h. CCD lumen is completely occluded at A, B and D groups from 2 h, 24 h, 4 days to 7 days. However, CCA lumen is occluded at 2 h but progressively recanalized from 24 h, 4 days to 7 days at C and E groups. A  LED irradiation alone (LED group), B LED irradiation plus intravenous bolus injection of 0.45 mg/kg rt-PA alone (0.45 mg/kg rt-PA group), C  LED irradiation plus intravenous bolus injection of 0.9 mg/kg rt-PA alone (0.9 mg/kg rt-PA group), D LED irradiation plus intravenous bolus injection of 1 × 10⁶ iPSCs alone (iPSCs group), E LED irradiation plus intravenous bolus injection of 0.45 mg/kg rt-PA and 1 h later, bolus injection of 1 × 10⁶ iPSCs (0.45 mg/kg rt-PA plus iPSCs group). LED = light-emitting diode; n = 8 in each group. Arrow indicates carotid bifurcation. Arrowhead and dot-line indicate the stenosis segment. h = hour; d = day. Scale bar in A (2 h) = 1 mm

Fig. 3

Single immunoconfocal localization of DAPI (blue color) and SSEA1 (red color) with ET-1, ICAM-1, IL-1 beta, AP-2, BDNF, and VEGF (green color) in carotid artery wall. DAPI can be seen before LED irradiation and at 1 day and 7 days after LED irradiation. SSEA1 is not visible before LED irradiation and at 1 day and 7 days in LED alone group, but is strongly visible at 1 day and 7 days after iPSC treatment. ET-1, ICAM-1, and IL-1 beta are rarely seen in CCA artery wall before LED irradiation. However, ET-1, ICAM-1, and IL-1 beta are stained strongly at 1 day and 7 days in LED alone group, but their immunofluorescence is reduced after iPSCs treatment. AP-2, BDNF and VEGF are mildly expressed in CCA artery wall before LED irradiation. However, the expressions of AP-2, BDNF and VEGF are reduced in LED alone group at 1 day and 7 days. After iPSCs treatment, the expressions of AP-2, BDNF and VEGF are increased. AP-2 = angiopoietin-2; BDNF = brain-derived neurotrophic factor; DAPI = 4’,6-diamidino-2-phenylindole; ET-1 = endothelin-1; ICAM-1 = intercellular adhesion molecule-1; IL-1 = interleukin 1 beta; iPSCs = mouse induced pleuripotent stem cells; LED = light-emitting diode; SSEA1 = stage-specific embryonic antigen 1; VEGF = vascular endothelial growth factor

Fig. 4

Statistical results of the optical density of single immunoconfocal staining of SSEA1, ET-1, ICAM-1, IL-1 beta, AP-2, BDNF and VEGF. One-way analysis of variance with Tukey–Kramer test for post-hoc comparisons is used for multiple comparison of means. *P < 0.05, ^#P < 0.01

Fig. 5

Immunoconfocal colocalization of SSEA1 (red color) with DAPI (blue color), and ET-1, ICAM-1, IL-1 beta, AP-2, BDNF, and VEGF (green color) in CCA artery wall. SSEA1 is not visible in the groups of before LED and LED alone but is strongly expressed after iPSCs treatment at 1 day and 7 days. ET-1 and IL-1 beta are rarely expressed before LED irradiation. However, they show strong expressions in LED alone group at 1 day and 7 days, and the expressions are reduced after iPSCs treatment. AP-2, BDNF and VEGF are lightly visible in before LED group and reduced in LED alone group at 1 day and 7 days. However, AP-2, BDNF and VEGF are increased after iPSCs treatment. ICAM-1 is not colocalized with SSEA1 due to the conflict of secondary antibodies. AP-2 = angiopoietin-2; BDNF = brain-derived neurotrophic factor; DAPI = 4’,6-diamidino-2-phenylindole; ET-1 = endothelin-1; ICAM-1 = intercellular adhesion molecule-1; IL-1 = interleukin 1 beta; iPSCs = mouse induced pleuripotent stem cells; LED = light-emitting diode; SSEA1 = stage-specific embryonic antigen 1; VEGF = vascular endothelial growth factor

Fig. 6

Immunoconfocal colocalization of iRFP⁺-iPSCs and DAPI with AP-2, BDNF or VEGF. AP-2, BDNF and VEGF are colocalized with DAPI in iRFP⁺-iPSCs. AP-2 = angiopoietin-2; BDNF = brain-derived neurotrophic factor; DAPI = 4’,6-diamidino-2-phenylindole; iRFP = near-infrared fluorescent protein; VEGF = vascular endothelial growth factor

Fig. 7

Hematoxylin and eosin (H&E) and immunoconfocal stain of heart, kidney, liver and lung at 28 days after intravenous iPSC treatment. H&E stain shows no abnormal cells can be found and immunoconfocal stain shows SSEA1-positive immunoreactivity is not seen in heart, kidney, liver and lung compared to normal. DAPI = 4’,6-diamidino-2-phenylindole; iPSCs = mouse induced pleuripotent stem cells; SSEA1 = stage-specific embryonic antigen 1