**1. Introduction**

Cardiovascular diseases are still one of the most common causes of morbidity and mortality worldwide. In this regard, atherosclerosis—a chronic inflammatory disease of the arteries—has long been identified as the underlying cause that could ultimately lead to fatal events such as myocardial infarction, strokes [1] and also to peripheral artery disease (PAD) [2]. Atherosclerosis is characterized as a progressing process of plaque growth in the arterial vessel wall that develops in the setting of hyperlipidemia and goes along with vascular lumen stenosis, plaque rupture and erosion [3]. The growth of pre-existing collateral arteries (also termed as arteriogenesis) represents an endogenous mechanism of bypassing occluded vessels and is an important adaptive response to maintain or restore arterial perfusion [4]. Arteriogenesis occurs in tissues near to arterial stenosis whereas down-stream ischemic regions undergo angiogenesis, which is the growth of new capillaries. Collateral growth is driven by hemodynamic forces such as shear stress [5,6] and wall stress and leads to initial vasodilation

due to increased levels of nitric oxide [7]. It is the reason why significant stenoses of main arteries may remain asymptomatic in patients for some time. However, in most cases, collateral growth could not ensure su fficient blood supply to the a ffected region, which becomes ischemic over time. Therefore, developing therapeutic approaches to improve this process is certainly desirable.

Just like atherosclerosis, collateral growth is critically driven by inflammatory processes. Chemokines, such as CC-chemokine ligands (CCL)2, and adhesion molecules, such as intercellular adhesion molecules (ICAM)-1, mediate the recruitment and accumulation of mainly monocytes into the arterial wall at sites of collateral growth. The proliferation of endothelial cells and smooth muscle cells subsequently lead to the lumen size expansion of the a ffected collateral artery [8]. In recent years, we have successfully used the Toll-like receptor (TLRs) 2/6 agonist macrophage activating protein of 2-kDA (MALP-2) to boost inflammatory processes and promote adaptive and regenerative mechanisms. TLRs belong to the class of pattern recognition receptors which were initially discovered on mammalian immune cells and recognize conserved pathogen-associated molecular patterns in order to initiate the immune response and combat bacterial infections [9]. In addition, an important role of TLRs has emerged later in many physiological as well as pathophysiological processes. For example, during atherogenesis, pattern recognition receptors such as TLRs are involved in the induction of inflammatory processes in response to exogenous and endogenous ligands which arise after necrotic cell death or extracellular matrix degradation [10]. MALP-2 is a common diacylated bacterial lipopeptide which is recognized by a heterodimer of TLR2 and TLR6 and was originally described as a potent activator of macrophages [11–13]. We recently reported that a single application of MALP-2 triggers beneficial vascular e ffects such as angiogenesis [14], endothelial wound healing and the inhibition of neointima formation following vascular injury [15]. Additionally, we observed the augmented angiogenic potential of mesenchymal stem cells after MALP-2 treatment in a sheep model of tissue engineering [16]. Given the importance of inflammatory processes for collateral growth—and because we had already established vascular cells as suitable target cells for MALP-2—we next investigated the potential of MALP-2 to promote blood flow recovery after the experimental ligation of the femoral artery by collateral growth in mice.

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

#### *2.1. Reagents and Antibodies*

The macrophage-activating lipopeptide of 2 kDa (MALP-2) was synthesized and purified as described before [11]. Fibronectin was purchased from Promocell (Heidelberg, Germany), calcein-AM from eBioscience (San Diego, CA, USA), 4-,6-diamidino-2-phenylindole (DAPI) from Sigma-Aldrich (Munich, Germany). Phenylephrine (PE), acetylcholine (ACh), noradrenaline and N-Nitroarginine methyl ester (L-NAME) were purchased from Sigma-Aldrich. Indomethacin was obtained from Alfa Aaesar (Thermo Fisher Scientific, Waltham, MA, USA), sodium nitroprusside from Honeywell (Seelze, Germany) and U46619 from Cayman Chemical (Ann Arbor, MI, USA). Antibodies for immunofluorescence against CD68, CD31 and Ki67 were from Abcam (Cambridge, UK) and against α-SMA-Cy3 were from Sigma-Aldrich. Antibodies for Western blot against VCAM-1 and β-Actin were from Santa Cruz (Dallas, TX, USA) and against p-AKT (S473), AKT, p-eNOS (S1177) and eNOS were from Cell Signaling Technology (Danvers, MA, USA). Appropriate secondary antibodies for immunofluorescence and Western blot were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

#### *2.2. Mice and Cells*

The animal handling and all experimental procedures were in accordance with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and were approved by the Animal Care and Use Committee of the state Hessen (approval reference numbers V54-19c20/15-B2/1152 (23.05.17); B2-1077 (29.07.16)). For femoral artery ligation (FAL), 8–12-week-old male C57BL/6 and BALB/c mice were purchased from Charles River (Sulzfeld, Germany). Six to ten-week-old male Apoe-deficient mice with a C57BL/6 background from our own breeding were fed a high fat diet (HFD, 21% butterfat, 1.5% cholesterol, Ssniff, Soest, Germany) for 12 weeks and operated on thereafter. Adductor muscles were isolated from the left and right upper hind limbs of 10-week-old male C57BL/6 mice, cut into 1–2 mm pieces with fine scissors and 4 pieces were placed in a well of a 96-well plate for ex vivo stimulation with MALP-2. The endothelial MyEnd cell line was grown in Dulbecco's modified Eagle medium (DMEM, Gibco, Darmstadt, Germany) with 10% fetal calf serum (FCS, PAN-Biotech, Aidenbach, Germany) and 1% penicillin/streptomycin (100 U/mL and 100 mg/mL, Sigma-Aldrich). The MyEnd cells showed typical endothelial properties and, as they grew to complete confluence, were highly positive for the endothelial marker CD31 and expressed the MALP-2 receptors TLR2 and TLR6 (Figure S1). The monocyte/macrophage cell line J774A.1 was grown in DMEM-Glutamax (Gibco) with 10% FCS and 1% penicillin/streptomycin (P/S).

#### *2.3. Experimental Femoral Artery Ligation (FAL)*

The mice were subjected to FAL as described elsewhere [17]. During the surgical procedure, the mice were under general anesthesia with isoflurane (2.5% for induction, 1.5–2.0% maintenance). After the FAL, the mice were intravenously injected with MALP-2 (1 μg in 125 μL phosphate-buffered saline (PBS) per mouse) or vehicle control (125 μL PBS). For postoperative analgesia, carprofen (5 mg/kg body weight) was subcutaneously injected once prior to surgery. The contralateral leg served as the control. After the termination of experiments, the mice were euthanized by an anesthetic overdose.

#### *2.4. Laser Speckle Imaging*

The perfusion of the hind paws was assessed using a laser speckle imaging device (moorFLPI-2; software for acquisition and MoorFLPI Review V5.0 for evaluation, Moor Instruments, Axminster, UK) on a heating plate (37 ◦C) before the FAL (d0 pre), immediately after (d0 post), and d3, d7 and d10 after the FAL.

#### *2.5. Histology and Immunohistochemistry*

The mice were perfused with 10 mL of a vasodilation buffer (100 μg adenosine, 1 μg sodium nitroprusside, 0.05% bovine serum albumin in PBS, pH 7.4), followed by 10 mL of 3% paraformaldehyde post mortem. Tissue from the ligated left and the not ligated right adductor muscles was harvested and placed in 15% sucrose in PBS for 4 h and overnight at 4 ◦C in 30% sucrose in PBS. The tissue was cryopreserved in Tissuetek (Sakura Finetek, Staufen, Germany) and cut into 8 μm cryosections. A morphometric analysis was performed using haematoxilin-eosin staining to evaluate the dimensions of the collateral arteries with the help of ImageJ software (National Institutes of Health, Bethesda, MD, USA). The cryosections were fixed with 5% paraformaldehyde and stained with antibodies against Ki-67, CD31, α-SMA or CD68. The slides were covered with Mowiol (Sigma-Aldrich) and analyzed with a confocal microscope (Leica SP5, Leica, Wetzlar, Germany).

#### *2.6. Organ Chamber Experiments (Wire Myography)*

The male C57BL/6 mice of 10–12 weeks were killed by CO2/O2 inhalation. The mesenteric artery was dissected free from surrounding fat and connective tissue and directly mounted in a wire myograph (Danish Myo Technology, Aarhus, Denmark) containing Krebs solution (119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2·2H2O, 1.17 mM MgSO4·7H2O, 20 mM NaHCO3, 1.18 mM KH2PO4, 0.027 mM EDTA, 11 mM glucose). Mesenteric arterial segments (2 mm) were distended to the diameter at which maximal contractile responses to 10 μM noradrenaline could be obtained. The maximal relaxing response to acetylcholine (ACh, 10 μM) was recorded during a contraction induced by 10 μM noradrenaline; arterial segments which showed less than 85% relaxation were discarded from the experiments.

#### *2.7. Real-Time PCR*

For the analysis of the mRNA expression, the total RNA was isolated using RNA-Solv®Reagent (Omega Bio-tek, Norcross, GA, USA) following the manufacturer's instructions and reverse-transcribed with SuperScript reverse transcriptase, oligo(dT) primers (Thermo Fisher Scientific), and deoxynucleoside triphosphates (Promega, Mannheim, Germany). Real-time PCR was performed in duplicates in a total volume of 20 μL using Power SYBR green PCR Master Mix (Thermo Fisher Scientific) on a Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) in 96-well PCR plates (Applied Biosystems). The SYBR Green fluorescence emissions were monitored after each cycle. For normalization, the expression of glyceraldehyde 3-phosphate dehydrogenase as housekeeper was determined in duplicates. The gene expression was calculated using the 2− ΔΔCt method. The PCR primers were obtained from Microsynth AG (Balgach, Switzerland) and are available upon request.

#### *2.8. Enzyme-Linked Immunosorbent Assay (ELISA)*

The supernatant from cultured tissue pieces of the adductor muscles of C57BL/6 mice was analyzed for CCL2, GM-CSF, IL-1α and TNF-α using a mouse-specific ELISA from R&D Systems (Minneapolis, MN, USA) according to the manufacturer's protocol with the help of an Infinite M200 PRO plate reader (TECAN Instruments, Maennedorf, Switzerland).

#### *2.9. Western Blot*

The total protein was extracted with a buffer that contained 150 mM NaCl, 1% Triton X-100, 0.5% sodiumdeoxycholate, 0.1% SDS and 50 mM Tris that was supplemented with a protease inhibitor cocktail (Roche, Penzberg, Germany). The total protein content was measured using a protein quantitation assay (Thermo Fisher Scientific) according to the manufacturer's protocol. The total protein (20 μg) was loaded onto 10% denaturing SDS gel and transferred to 0.45 mm polyvinylidene fluoride membranes (GE Healthcare, Little Chalfont, UK) for immunoblotting. The membranes were blocked with 5% nonfat dry milk (Sigma-Aldrich) and probed with primary antibodies against VCAM-1, β-Actin, p-AKT, AKT, p-eNOS and eNOS, followed by horseradish peroxidase–labeled secondary antibodies. Proteins were detected using a chemiluminescence substrate (Bio-Rad Laboratories, Hercules, USA). The results were documented on a Chemo-star imaging system (INTAS, Göttingen, Germany). The signal intensity of the chemiluminescence was quantified using Quantity One software (Bio-Rad).

#### *2.10. Griess Assay*

The MyEnd cells were plated in fibronectin-coated wells of a 96-well plate (TPP, Trasadingen, Switzerland) in DMEM with 10% FCS and 1% P/S and grown to complete confluence. The cells were starved in DMEM with 1% FCS and 1% P/S for 16 h and stimulated with MALP-2 (1 μg/mL) for 2 h. The NO levels in each well were measured using a Griess reagen<sup>t</sup> (Sigma-Aldrich) according to the manufacturer's instructions.

#### *2.11. Adhesion Assay*

The MyEnd cells were plated in fibronectin-coated wells of a 48-well plate (TPP) in DMEM with 10% FCS and 1% P/S and grown to complete confluence. The cells were starved in DMEM with 1% FCS and 1% P/S for 16 h and stimulated with MALP-2 (1 μg/mL) for 6 h. In parallel, J774A.1 cells were labeled with 5 μM of calcein-AM (Invitrogen, Carlsbad, CA, USA). according to the manufacturer's instructions. After stimulation, the MyEnd cells were washed twice with 500 μL of PBS per well; 0.5×10<sup>6</sup> labeled J774A.1 cells in 500 μL of DMEM with 1% FCS were added per well and co-cultured for 1 h in 5% CO2 at 37 ◦C. After co-incubation, each well was washed three times with 500 μL of PBS and 10 high powerfield (HPF) digital images were taken using an Axio Vert.A1 microscope

equipped with an AxioCam MRm camera (Carl Zeiss, Microimaging, Jena, Germany). The adhered calcein-AM-labeled J774A.1 cells per HPF image were counted using ImageJ software.

#### *2.12. Statistical Analysis*

All the data are represented as means ± SEM. The data were compared using the 2-tailed Student t-test for independent samples or by a 1-way ANOVA followed by the Tukey multiple comparison test (GraphPad Prism, version 6.05; GraphPad Software, La Jolla, CA, USA). A value of *P* < 0.05 was considered statistically significant. The numbers of independent experiments are indicated in each figure legend. The real-time PCR was performed in technical duplicates.
