Stimulation of vascular smooth muscle cell proliferation by stiff matrix via the IKCa channel-dependent Ca2+ signaling.

Vascular stiffening, an early and common characteristic of cardiovascular diseases (CVDs), stimulates vascular smooth muscle cell (VSMC) proliferation which reciprocally accelerates the progression of CVDs. However, the mechanisms by which extracellular matrix stiffness accompanying vascular stiffening regulates VSMC proliferation remain largely unknown. In the present study, we examined the role of the intermediate-conductance Ca2+ -activated K+  (IKCa ) channel in the matrix stiffness regulation of VSMC proliferation by growing A7r5 cells on soft and stiff polydimethylsiloxane substrates with stiffness close to these of arteries under physiological and pathological conditions, respectively. Stiff substrates stimulated cell proliferation and upregulated the expression of the IKCa channel. Stiff substrate-induced cell proliferation was suppressed by pharmacological inhibition using TRAM34, an IKCa channel blocker, or genetic depletion of the IKCa channel. In addition, stiff substrate-induced cell proliferation was also suppressed by reducing extracellular Ca2+ concentration using EGTA or intracellular Ca2+ concentration using BAPTA-AM. Moreover, stiff substrate induced activation of extracellular signal-regulated kinases (ERKs), which was inhibited by treatment with TRAM34 or BAPTA-AM. Stiff substrate-induced cell proliferation was suppressed by treatment with PD98059, an ERK inhibitor. Taken together, these results show that substrates with pathologically relevant stiffness upregulate the IKCa channel expression to enhance intracellular Ca2+ signaling and subsequent activation of the ERK signal pathway to drive cell proliferation. These findings provide a novel mechanism by which vascular stiffening regulates VSMC function.

Electrospun PELCL membranes loaded with QK peptide for enhancement of vascular endothelial cell growth.

One of the major challenges in tissue engineering of small-diameter vascular grafts is to inhibit intimal hyperplasia and keep long-term patency after implantation. Rapid endothelialization of the grafts could be an effective approach. In this study, QK, a peptide mimicking vascular endothelial growth factor, was selected as the bioactive substrate and loaded in electrospun membranes for enhancement of vascular endothelial cell growth. In detail, QK peptide was firstly introduced with poly(ethylene glycol) diacrylate into a thiolated chitosan solution that could transfer into hydrogel. Then, suspensions or emulsions of poly(ethylene glycol)-b-poly(L-lactide-co-ε-caprolactone) (PELCL) containing QK peptide (with or without chitosan hydrogel) were electrospun into fibrous membranes. For comparison, the electrospun PELCL membrane without QK was also fabricated. Results of release behaviors showed that the electrospun membranes, especially that contained chitosan hydrogel prepared by suspension electrospinning, could successfully encapsulate QK peptide and maintain its secondary structure after released. In vitro cell culture studies exhibited that the release of QK peptide could accelerate the proliferation of vascular endothelial cells in the 9 days. It was suggested that the electrospun PELCL membranes loaded with QK peptide might have potential applications in vascular tissue engineering.

Targeted delivery of microRNA-126 to vascular endothelial cells via REDV peptide modified PEG-trimethyl chitosan.

Manipulation of gene expression by means of microRNAs (miRNAs) is one of the emerging strategies to treat cardiovascular and cancer diseases. Nevertheless, efficient delivery of miRNAs to a specific vascular tissue is limited. In this work, a short peptide Arg-Glu-Asp-Val (REDV) was linked to trimethyl chitosan (TMC) via a bifunctional poly(ethylene glycol) (PEG) linker for the targeted delivery of microRNA-126 (miRNA-126) to vascular endothelial cells (VECs). The morphology, serum stability and cytotoxicity of the polyplex/miRNA complexes, namely, TMC/miRNA, TMC-g-PEG/miRNA and TMC-g-PEG-REDV/miRNA, were investigated along with the cellular uptake, proliferation and in vitro miRNA transfection efficiency. By REDV modification, the TMC-g-PEG-REDV/miRNA complex showed negligible cytotoxicity, increased expression of miRNA-126 and enhanced VEC proliferation compared with the TMC/miRNA and TMC-g-PEG/miRNA complexes. In particular, the approaches adopted for the miRNA delivery and targeted peptide REDV modification promote the selective uptake and the growth of VECs over vascular smooth muscle cells. It was suggested that the REDV peptide-modified TMC-g-PEG polyplex could be potentially used as a miRNA carrier in artificial blood vessels for rapid endothelialization.

Peptide-modified PELCL electrospun membranes for regulation of vascular endothelial cells.

The efficiency of biomaterials used in small vascular repair depends greatly on their ability to interact with vascular endothelial cells (VECs). Rapid endothelialization of the vascular grafts is a promising way to prevent thrombosis and intimal hyperplasia. In this work, modification of electrospun membranes of poly(ethylene glycol)-b-poly(l-lactide-co-ε-caprolactone) (PELCL) by three different peptides for regulation of VECs were studied in order to obtain ideal bioactive biomaterials as small diameter vascular grafts. QK (a mimetic peptide to vascular endothelial growth factor), Arg-Glu-Asp-Val (REDV, a specific adhesive peptide to VECs) and Val-Ala-Pro-Gly (VAPG, a specific adhesive peptide to vascular smooth muscle cells) were investigated. Surface properties of the modified membranes and the response of VECs were verified. It was found that protein adsorption and platelet adhesion were effectively suppressed with the introduction of QK, REDV or VAPG peptides on the PELCL electrospun membranes. Both QK- and REDV-modified electrospun membranes could accelerate the proliferation of VECs in the first 9days, and the QK-modified electrospun membrane promoted cell proliferation more significantly than the REDV-modified one. The REDV-modified PELCL membrane was the most favorable for VECs adhesion than QK- and VAPG-modified membranes. It was suggested that QK- or REDV-modified PELCL electrospun membranes may have great potential applications in cardiovascular biomaterials for rapid endothelialization in situ.

Nanofiber-mediated microRNA-126 delivery to vascular endothelial cells for blood vessel regeneration.

UNLABELLED: As manipulation of gene expression by virtue of microRNAs (miRNAs) is one of the emerging strategies for cardiovascular disease remedy, local delivery of miRNAs to a specific vascular tissue is challenging. In this work, we developed an efficient delivery system composed of electrospun fibrous membranes and target carriers for the intracellular delivery of miRNA-126 (miR-126) to vascular endothelial cells (VECs) in the local specific vascular environment. A bilayer vascular scaffold was specially prepared via emulsion electrospinning of poly(ethylene glycol)-b-poly(l-lactide-co-ε-caprolactone) (PELCL) and dual-power electrospinning of poly(ε-caprolactone) (PCL) and gelatin. The inner layer of PELCL, which was loaded with complexes of miR-126 in REDV peptide-modified trimethyl chitosan-g-poly(ethylene glycol), regulated the response of VECs, while the outer layer of PCL/gelatin contributed to the mechanical stability. Biological activities of the miR-126-loaded electrospun membranes were evaluated by cell proliferation and SPRED-1 expression of a miR-126 target gene. By encapsulating targeting complexes of miR-126 in the electrospun membranes, a sustained release profile of miRNA was obtained for 56days. Significant down-regulation of SPRED-1 gene expression in VECs was detected on day 3, and it was found that miR-126 released from the electrospun membranes accelerated VEC proliferation in the first 9days. The bilayer vascular scaffold loaded with miR-126 complexes could also improve endothelialization in vivo. These results demonstrated the potential of this approach towards a new and more effective delivering system for local delivery of miRNAs to facilitate blood vessel regeneration. STATEMENT OF SIGNIFICANCE: Tissue engineering of small-diameter blood vessels is still challenging because of thrombosis and low long-term patency. The manipulation of gene expression by miRNAs could be a novel strategy in vascular regeneration. Here, we report an efficient delivery system of electrospun fibrous scaffold combined with REDV peptide-modified trimethyl chitosan for targeted intracellular delivery of miR-126 to VECs in the local vascular environment. Results exhibited that miR-126 released from the electrospun membrane could modulate VEC proliferation via down-regulation of SPRED-1 gene expression. The electrospun scaffolds loaded with target-delivery carriers may serve as an ideal platform for local delivery of miRNAs in the vascular tissue engineering.