Use of kv1.3 channel blockers for the prevention of restenosis in human vessels: mechanisms and outcomes in diabetic patients
- Autores: Marycarmen Arévalo Martínez
- Directores de la Tesis: Teresa Pérez García (dir. tes.), Pilar Cidad Velasco (dir. tes.)
- Lectura: En la Universidad de Valladolid ( España ) en 2020
- Idioma: español
- Tribunal Calificador de la Tesis: Irene Cozar Castellano (presid.), A.P. Dantas (secret.), Iain Andrew Greenwood (voc.)
- Programa de doctorado: Programa de Doctorado en Investigación Biomédica por la Universidad de Valladolid
- Materias:
- Enlaces
- Tesis en acceso abierto en: UVADOC
- Resumen
Vascular smooth muscle cells (VSMCs) can undergo phenotypic modulation (PM) to a dedifferentiated state, which contributes to angiogenesis and vessel repair. PM is triggered by vascular surgeries such as those directed to unclog obstructed vessels. However, an excessive VSMC migration and proliferation drives intimal hyperplasia (IH) leading to restenosis. This situation is even worse in patients with background diseases like type 2 diabetes mellitus (T2DM). T2DM patients have more aggressive forms of vascular disease and worse outcomes, with exacerbated restenosis after vascular surgery.
We have previously demonstrated that an increased functional expression of the potassium channel Kv1.3 contributes to PM in several models of VSMCs, as Kv1.3 blockers inhibit VSMCs migration and proliferation. In addition, we found that Kv1.3 increased activity upon PM is a consequence of Kv1.5 downregulation, so that the changes in Kv1.3 to Kv1.5 ratio can define VSMCs phenotype.
Objective: Here, we sought to explore the efficacy of Kv1.3 inhibition for the prevention of remodeling in human vessels and the mechanism linking the switch of Kv1.3/Kv1.5 ratio to PM. In addition, we investigated if the augmented remodeling observed in T2DM vessels is associated to an increased Kv1.3 activity, in order to determine if the use of Kv1.3 blockers could improve the outcomes of vascular surgery these vulnerable groups.
Approach: Vascular remodeling was explored in vessel rings in organ culture obtained from human mammary artery (hMA), human saphenous vein (hSV) and human renal artery (hRA) samples. Vessels were incubated for 2 weeks in organ culture. We studied the effects of Kv1.3 blockers (PAP-1, margatoxin), or Kv1.5 overexpression on IH development. In addition, primary VSMCs culture were used to explore the effects of these maneuvers on migration and proliferation.
An in-vivo model of vascular disease was developed by feeding hypertensive mice with high fact diet (BPH/HFD). These mice developed obesity, glucose intolerance, insulin resistance and hypertension. Carotid ligation was used to get an in-vivo model of IH in control (BPH) and diseased (BPH/HFD) vessels.
IH in both, human and mouse vessel samples was explored with hystomorphometric analysis of 7-m sections stained with Masson trichrome. In addition, we studied gene and miRNA expression by qPCR and protein expression by western blot and immunohistochemistry.
Results: Kv1.3 blockade prevented IH induced by 20% FBS in our in-vitro intimal hyperplasia model by inhibiting proliferation, migration, and extracellular matrix secretion. The effects of Kv1.3 blockers were reproduced by Kv1.5 overexpression, being both effects non-additive.
Myocardin knock-down in human vessels in organ culture reduced Kv1.5 expression and induced IH, while Kv1.5 overexpression inhibited IH without affecting myocardin expression. Moreover, myocardin overexpression upregulated Kv1.5 expression in VSMCs.
When human samples were categorized as T2DM or non-T2DM, we found that T2DM vessels showed increased IH in organ culture. Also, T2DM VSMCs had higher rates of proliferation and migration than non-T2DM cells. We observed a higher expression of Kv1.3 in T2DM vessels, and consequently PAP-1 was more efficient preventing vascular remodeling in T2DM vessels. Differences in T2DM samples persisted in VSMCs cultures, suggesting a metabolic memory of those cells. We carried out a miRNA PCR array to explore the contribution of miRNAs to T2DM epigenetic signature in human VSMCs. We focused on miR-126, which was upregulated in T2DM VSMC in proliferative phenotype. miR-126 overexpression in non-T2DM increased migration and proliferation rate, but had no effect on T2DM VSMCs.
Finally, in our BPH/HFD animal model, we found increased expression of Kv1.3 in VSMCs and an increased vascular remodeling upon carotid ligation compared to control (BPH) mice. Treatment with PAP-1 prevented IH upon carotid ligation in both groups, and decreased obesity and improved insulin sensitivity in BPH/HFD.
Conclusions: Our data indicated that Kv1.5 channel is a myocardin-regulated gene in human VSMCs. Kv1.5 downregulation upon PM leaves Kv1.3 as the dominant Kv1 channel expressed, increasing Kv1.3 functional contribution to dedifferentiated phenotype. Kv1.3 channel blockade prevents vascular remodeling of human vessels in organ culture by inhibiting VSMCs proliferation, migration and extracellular matrix secretion, and Kv1.5 overexpression also inhibits vascular remodeling by occluding the K1.3 channel effects.
Kv1.3 blockade is more efficient in T2DM vessels reducing IH in organ culture than in those from non-T2DM patients, as T2DM vessels show higher expression of Kv1.3 channels and increased remodeling. Epigenetic changes in VSMC from T2DM associated to differences in miRNA profile. Our data indicated that the increase of miR-126 in T2DM VSMCs cultures contributes to the metabolic memory of these cells.
BPH/HFD is an animal model that parallels the characteristics of T2DM human vessels: increased Kv1.3 channel expression and vascular remodeling. Kv1.3 blockers not only showed higher efficiency in the prevention of IH in diseased vessels compared to healthy vessels, but also ameliorate metabolic risk factors in the BPH/HFD model.
