**1. Introduction**

Arteriogenesis, which is defined as the growth of pre-existing arteriolar connections into functional arteries compensating for the loss of an artery due to occlusion [1], particularly involves the proliferation of vascular endothelial cells (ECs) and smooth muscle cells (SMCs). The driving force for arteriogenesis is increased fluid shear stress [2,3]. This mechanical force, which can be sensed directly by ECs, has recently been shown to be linked to local activation of collateral ECs. By mediating the release of extracellular RNA (eRNA) from ECs, eRNA promotes the binding of vascular endothelial growth factor A (VEGFA) to VEGF receptor 2 (VEGFR2) [4], thereby promoting local vascular EC proliferation as well as activation of a mechanosensory complex, consisting of VEGFR2, platelet endothelial cell

adhesion molecule 1 (PECAM-1), and vascular endothelial cell cadherin (VE-cadherin) [5], triggering collateral artery growth.

SMCs, located at the abluminal side, are not able to sense shear stress, and relatively little is known about the mechanisms triggering SMC proliferation in arteriogenesis. Growth factors, such as fibroblast growth factor 2 (FGF-2) and platelet-derived growth factor BB (PDGF-BB) have been shown to be important for SMC proliferation in arteriogenesis [6,7] (and own unpublished results of M. Lasch). This might in particular be related to their function to induce the expression of early growth response 1 (*Egr1*) [8], a transcriptional regulator, which has been shown to control cell cycle progression in arteriogenesis [9]. Interestingly, it has been demonstrated that the receptor for FGF-2, namely FGFR-1, which is expressed on collateral SMCs but not on ECs, is increased expressed only during a short time frame after induction of arteriogenesis [10]. These data indicate that the point in time of FGF-2 application is critical and might explain the outcome of clinical studies in which FGF-2 treatment showed limited effects in patients with vascular occlusive diseases [11,12].

The proliferation of vascular SMCs in arteriogenesis is characterized by the transition from the contractile to the synthetic (proliferating) SMC type. This process is associated with reduced mRNA and protein levels of contractile genes such as α-smooth muscle actin (αSM-actin) and paralleled by an increased expression of *Egr1*. [9,13,14]. The opposed gene expression of contractile genes and *Egr1*, which is tightly regulated by the transcriptional co-activators myocardin and myocardin-related transcription factors (MRTFs) and the ternary complex factor ETS like protein Elk-1, which compete for the binding to the transcription factor serum response factor (SRF) [15–17], has been demonstrated for the process of arteriogenesis by our group [9].

The potassium channels KV1.3 and KCa3.1 have been shown to be involved in cell cycle regulation by activating intracellular signaling pathways [18,19], and to play a role in modulating vascular SMC proliferation [20,21]. Since the mechanisms triggering vascular SMC proliferation in arteriogenesis are still not very well described, we decided to investigate the functional contribution of KV1.3 and KCa3.1 to the proliferation of vascular SMCs in collateral artery growth. The mode of action of these potassium channels in terms of proliferation is still under debate and several mechanisms, either ion flux dependent or independent, have been proposed [18,19]. Both potassium channels, KV1.3 and KCa3.1, have been demonstrated to be upregulated in proliferating SMCs, whereby their specific blockade interfered with cell cycle progression. Increased expression levels of KV1.3 have been detected in vitro in proliferating SMCs isolated from murine femoral arteries or from human donors [21,22] as well as under pathological situations such as neointima hyperplasia in vivo [22,23]. Moreover, it has been shown that blockade of KV1.3 with selective blockers such as 5-(4-phenoxybutoxy)psoralen (PAP-1) inhibited migration and proliferation of SMCs in vitro [22,23] and in vivo [23,24]. Similar to KV1.3, KCa3.1 has been found to be upregulated upon stimulation with PDGF-BB in proliferating SMCs in vitro [20] and in models of hyperplasia in vivo [25,26]. Blocking KCa3.1 with the selective blocker TRAM-34 in contrast interfered with SMC proliferation both, in vitro and in vivo [20,25,27,28]. Besides its effect on SMC proliferation, KCa3.1 plays a major role in endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilation as shown in KCa3.1 deficient mice [29].

In the present study we investigated the relevance of the potassium channels KV1.3 and KCa3.1 for SMC proliferation in growing collateral arteries by performing blocking studies employing the selective channel blockers PAP-1 and TRAM-34, respectively. From our results we conclude that KV1.3 contributes to SMC proliferation in arteriogenesis, whereas KCa3.1 is more likely to be involved in vasodilation.

#### **2. Materials and Methods**

#### *2.1. Animal Protocol and Treatments*

Male C57BL6/J mice, purchased from Charles River, were housed in cages and kept under 12 h day/night cycle with food and water ad libitum. All experiments were approved by the Bavarian Animal Care and Use Committee (ethical approval code ROB-55.2-1-54-2532-73-12 and

ROB-55.2Vet-2532.Vet\_02-17-99) and carried out according to the guidelines of the German law for protection of animal life. Mice at the age of 6 to 10 weeks were anesthetized with a combination of 0.5 mg/kg medetomidine (Pfister Pharma), 5 mg/kg midazolam (Ratiopharm GmbH), and 0.05 mg/kg fentanyl (CuraMED Pharma). Arteriogenesis was induced by right femoral artery ligation (FAL, occlusion (occ)), whereas the left femoral artery was sham operated (Figure 1) as previously described in [30].

**Figure 1.** Photographs of superficial collateral arteries in mouse adductor muscles. Photographs were taken 7 days after induction of arteriogenesis by femoral artery ligation (left picture) or sham operation (right picture). Mice were perfused with latex to better visualize collateral arteries. Pre-existing collaterals appear very fine and straight (arrows, right picture). Seven days after induction of arteriogenesis, grown collateral arteries show a typical corkscrew formation with increased vascular caliber size (arrows, left picture). Scale bar 5 mm

To block potassium channels, mice were treated either with the selective KV1.3 channel blocker (5-(4-phenoxybutoxy)psoralen (PAP-1, 40 mg/kg/d, intraperitoneally (i.p), Sigma-Aldrich) [31], or the selective KCa3.1 channel blocker TRAM-34 (120 mg/kg/d, i.p., Alomone Labs) [27], dissolved in peanut oil, at doses previously described [27,31]. The treatments started 4 h before the surgical procedure. Moreover, to uphold constant blood levels of the blockers, mice received two doses per day, one in the morning and one in the afternoon. When mice were treated with BrdU (Sigma-Aldrich), they received a single dose (1.25 mg/d dissolved in phosphate buffered saline (PBS), i.p.) starting directly after the surgical procedure.

#### *2.2. Laser Doppler Perfusion Measurements and Tissue Sampling*

The laser Doppler perfusion measurements were performed as described in [4]. In brief, hindlimb perfusion was measured using the laser Doppler imaging technique (Moor LDI2-IR, LDI 5061 and Moor Software 3.01, Moor Instruments) under temperature-controlled conditions (37 ◦C), and perfusion was calculated by right to left (occlusion (occ) to sham) flux ratios.

Prior to tissue sampling for (immuno-) histology, mice were perfused with an adenosine buffer (1% adenosine, 5% bovine serum albumin (BSA), both from Sigma-Aldrich dissolved in PBS, PAN Biotech, pH 7.4) for maximal vasodilation followed by perfusion with 3% paraformaldehyde (PFA, Merck) dissolved in PBS, pH 7.4, for cryoconservation, or 4% PFA for paraffin embedding [2]. For qRT-PCR analyses, mice were perfused with latex flexible compound (Chicago Latex) to visualize superficial collateral arteries (see also Figure 1) for dissection. After isolation, superficial collateral arteries were snap frozen on dry ice and stored at −80 ◦C until further investigations [9].

#### *2.3. Cell Culture*

Mouse primary artery smooth muscle cells (catalog number C57-6081, CellBiologics) were cultured in a SMC growth medium (SMCGM, CellBiologics) containing insulin and the growth factors fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) together with 20% fetal calf serum (FCS, PAN). For serum starvation, cells were cultured in Dulbecco's modified Eagle´s medium (DMEM, Thermo Fisher Scientific) with 1% FCS for 24 h. Thereafter, negative controls were stimulated with medium containing 2% FCS, positive controls with 10% FCS.

#### *2.4. Histology, Immunohistology, Proliferation Assay, and Immunocytochemistry*

Giemsa staining on paraffin fixed tissue samples was performed according to standard procedures, and slices were analyzed using an Axioskop 40 microscope (Carl Zeiss AG). BrdU staining of paraffin fixed tissue sections was performed with a BrdU detection kit (BD Pharmingen) according to the manufacturer´s procedure using the same microscope for evaluating the tissue sections.

To investigate the proliferation of mouse primary artery SMCs, a BrdU proliferation assay kit (Roche) was used according to the manufacturer´s instructions. In brief, mouse primary artery SMCs were seeded in a 96-well plate overnight, and after serum starvation in DMEM containing 1% FCS for 24 h, the mouse primary artery SMCs were cultured in DMEM with 10% FCS and treated with or without PAP-1 or TRAM-34, respectively, together with 10 mM BrdU. Cell proliferation was assessed by colorimetry with an Infinite F200 ELISA reader (TECAN).

For immunofluorescence staining, cryofixed tissue sections (10 μm) were stained with a rabbit anti-KV1.3 (catalog number APC-101) or a rabbit anti-KCa3.1 (catalog number APC-064) antibody (both from Alomone Labs) followed by a goa<sup>t</sup> anti-rabbit IgG Alexa fluor 488-conjugated antibody (catalog number 711-545-153, Jackson ImmunoResearch) together with a Cy3-conjugated mouse anti-αSM-actin antibody (catalog number C6198, Sigma-Aldrich) and an Alexa fluor 647-conjugated rat anti-CD31 antibody (catalog number 102515, BioLegend), followed by DAPI counter staining (catalog number 62248, Thermo Fisher Scientific). Images were taken with an Axio Imager 2 fluorescence microscope equipped with and an Axion ICc 5 camera and Axiovert software (Carl Zeiss) or using a LSM 880 confocal laser scanning microscope equipped with an Airycan module (Carl Zeiss) with ZEN black software for imaging acquisition. Imaging analysis of KV1.3 or KCa3.1 expression in αSM-actin positive SMCs or CD31 positive ECs was performed using the ZEN blue software. For the colocalization anaylsis, the ZEN colocalization tool was used (Carl Zeiss AG). The three-dimensional (3D) projection surface reconstruction of the images where done by using the Imaris software (Bitplane).

#### *2.5. RNA Isolation, cDNA Synthesis, and qRT-PCR*

The total RNA was isolated from the mouse primary artery SMCs or collateral arteries with Trizol (Life Technologies) and the residual DNA was removed by digestion with RQ1 RNase-Free DNase (Promega). Thereafter, RNA was purified with RNeasy MinElute columns (Qiagen) and reverse transcribed to cDNA using the QuantiTect® Reverse Transcription Kit (Qiagen) according to the manufacturer´s procedure. The qRT-PCR was performed as previously described [32] using the Power SYBR Green Kit (Life Technologies) and a StepOnePlus cycler (Life Technologies) and the following primers: 18S rRNA forward 5--GGACAGGATTGACAGATTGATAG-3-, reverse 5--CTCGTTCGTTATCGGAATTAAC-3-, αSM-actin forward 5--GAGCATCCGACACTGCTG-3-, reverse 5--GTACGTCCAGAGGCATAG-3-, fibroblast growth factor receptor-1 (*Fgfr1*) forward 5--CTTGCCGTATGTCCAGATCC-3-, reverse 5--TCCGTAGATGAAGCACCTCC-3-, platelet derived growth factor b (*Pdgfrb*) forward 5--AGGACAACCGTACCTTGGGTGACT-3-, reverse 5--CAGTTCTGACACGTACCGGGTCTC-3-, early growth response 1 (*Egr1*) forward 5--CGAACAACCCTATGAGCACCTG-3-, and reverse 5--CAGAGGAAGACGATGAAGCAGC-3-. To control specific amplification, melt curve analyses and agarose gels were performed. Data were analyzed using the ΔΔCt method [33] and results were normalized to the expression level of the 18S rRNA.

#### *2.6. Statistical Analyses*

Statistical analyses were performed using the GraphPad software PRISM6. All data are stated as means ± SEM. Results were tested for normality and statistical analyses were performed as specified in the figure legends. Results were considered to be statistically significant at *p* ≤ 0.05.
