Cuscuta chinensis Lamak (5 kg) was purchased from a local traditional medicine store (Jechoen, Korea) in July of 2017. The authenticity of the plant species was confirmed by one of the authors (Byoung Seob Ko; Korea Institute of Oriental Medicine), after which the seeds were ground into powder. Next, powder (4 kg) was extracted with 100% water (16 L) by heat reflux for 6 h, after which the supernatant was collected and dried using a freeze-dryer for 2 days. The extraction yield was of 0.005%, and 15 g of CCL-extract was used for subsequent experiments. The plants we used in the experiment were samples produced at GMP facilities in accordance with the Korean Food and Drug Administration's standards for manufacturing and quality control.

Female 6-week-old C57BL/6 mice were obtained from Dahan Biolink (Eumseong, South Korea) and divided into five-groups of seven. Mice were allowed to adapt to laboratory conditions (temperature: 20±2°C, relative humidity: 45±5%, light/dark cycle: 12 h) for 1 week. All animal experimental procedures were approved by the Ethics Committee of the Korea Institute of Oriental Medicine (approved no. 17-028). Mice were anesthetized by an animal anesthesia system (Vetequip, Abesko, Gyeonggi, Korea) using 2% isoflurane (Choongwae Pharma Corp., Seoul, Korea). OVX surgery was assessed according to the criteria of (11), and performed by liagation and excision of ovaries along the upper horns, through aseptic incisions of the dorsal skin and muscle layers. At 7 days after OVX surgery, mice were anesthetized for hind-limb ischemia (HLI) after which the vessels (arteries and veins) of the hind limb were excised on day 0, and surgery was modified according to the criteria of (12). After a 7 days recover period, 17β-estradiol (E2; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and CCL extract were prepared in 0.2% CMC (carboxymethyl cellulose) and administered for every 3-weeks by oral injection (E2 for 100 µg/kg/day and CCL for 150 and 450 mg/kg/day, respectively). Crude extract doses according to criteria of (13). Mice were euthanized 12 h after the final injection. And The liver, spleen, peritoneal fat, etc., including tissues from the surgical region, were collected and stored in 4% formalin or nitrogen immediately for long-term storage at −80°C for the purpose of analysis.

The blood flow rates in ischemic/non-ischemic (ISCH/nISCH) hind limbs were measured using a laser Doppler blood perfusion imager (PeriScan-PIM3; Perimed AB, Järfälla, Sweden) under anesthesia (Isoflurane; Choongwae Pharma Corp.) according to the manufacturer's recommendations. The rate of ISCH/nISCH blood flow was calculated at −7, 0, 7, 14, and 21 days after HLI by colorimetric assay using PIMSoft (v1.5; Perimed AB).

Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 5-µm-thick sections. Endogenous peroxidase and non-specific reactions were blocked with BLOXALL (for 30 min; Vector Lab. Inc., Burlingame, CA, USA) and CAS-BLOCK (for 1 h; Thermo Fisher Scientific, Pittsburgh, PA, USA) at room temperature (RT). Sections were subsequently incubated at RT for 4 h with an antibody against CD-31 (rabbit polyclonal, 1:250; Abcam). The sections were incubated with Dako REAL™ Envision™/HRP (Rabbit/Mouse) for 30 min at RT. Dako REAL™ DAB+ Chromogen (both from Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) was used as a chromogen, after which the sections were counterstained with Mayer's hematoxylin (Sigma-Aldrich; Merck KGaA) and mounted using Mounting Medium (Thermo Fisher Scientific). Tissue sample was examined under a light microscope (BX43; Olympus, Tokyo, Japan). And all images were captured using an Olympus DP-73 controller and cellSens standard (both from Olympus) under a microscope.

Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Hilden, Germany), and cDNA was synthesized from 1 µg of total RNAs using the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturers' instructions. Briefly, extracted RNA was used as a template for cDNA synthesis using Random Hexamer primers and RevertAid M-MuL V RT, incubated 5 min at 25°C followed by 60 min at 42°C. And termination of reaction by heating at 70°C for 5 min. Real-time PCR was performed using SYBR Premix EX Taq (Takara Bio Inc., Otsu, Japan) with 40 cycles for 30 sec at 95°C, for 30 sec at 60°C, and for 30 sec at 72°C by ABI PRISM 7500HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Quantification of target genes was accomplished by the comparative Ct method, and the levels of mRNAs were normalized by those of β-actin and presented as relative values. The primers used for real-time PCR were synthesized by Macrogen Inc. (Seoul, Korea) and are presented in Table I.

The levels of angiogenesis-related proteins and inflammatory cytokines were analyzed using a Mouse Angiogenesis Array Kit and Proteome Profiler Mouse Cytokine Array Kit (R&D System, Minneapolis, MN, USA) according to the manufacturer's instructions. Briefly, each membrane was incubated with the serum of each group overnight at 4°C. Bound protein was then detected with streptavidin conjugated to HRP using a digital image system (Chemiluminescence Imaging-Fusion SL, Analis, The Netherlands).

Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza (Walkersville, MD, USA) and maintained according to the manufacturer's instructions. Cell viability was detected using an EZ-Cytox kit (DoGenBio Co., Ltd., Seoul, Korea). The HUVEC cells were seeded at a density of 3×10⁴/well in 96-well plates. High sensitivity water soluble tetrazolium (WST) solution (10 µl per well in a 96-well plate) was added to the culture fluid at 1:1 ratio, while WST solution without cells was used as a blank control. Optical density was determined at various time-points (after 4 h) on a microtiter plate reader at 450 nm, after which the percentage viability was calculated using the following formula: Optical density of treated sample/optical density of untreated control ×100.

HUVECs migration was quantified using a corning Transwell system (Corning, NY, USA). HUVECs were seeded (2×10⁵/well) into the upper chamber (8 mm) of a Transwell plate in a serum-free medium. In the lower chamber, a complete medium was placed as a chemoattractant. Following incubation at 37°C under 5% CO₂ for 6 h, the cells on the top of the polycarbonate membrane were removed using cotton swabs, and the migrating cells on the lower side of the membrane were stained with hematoxylin and eosin (H&E) for 5 min. Four random fields were counted under the microscope during each assay.

HUVECs (5×10⁴ cells/well) were seeded on a Matrigel in 24-well plates supplemented with CCL (1, 10 and 100 µg/ml) and vascular endothelial growth factor (VEGF, 50 ng/ml), 17β-estradiol (E2, 1 nmol), and vinblasitne (1 pmol) were added to the media without the addition of growth factors. After 24 h, tubular structures of HUVECs were examined using an inverted microscope (Nikon, Melville, NY, USA), and the total tube length was measured in three fields (4×) using the ImageJ software version 1.51s (National Institutes of Health, Bethesda, MD, USA).

All the results presented are representative of at least 3 independent experiments. The results of the analyses of blood flow, capillary density, and gene expression are presented as the means ± SD. And the results of the analyses of cell viability, migration, and tube formation are shown as mean ± SD. Paired Student's t-tests were used to compare each group, while ANOVA with Tukey's test was used for multiple comparison tests using the PRISM software (v6.0; GraphPad, La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To determine whether CCL (150 or 450 mg/kg/day) treatment stimulates blood reperfusion in hind-limb ischemia, mice were treated with 17β-estradiol (E2) and CCL. As shown in Fig. 1, blood flow in the ischemic hind limbs decreased equally in all surgery groups immediately following ischemic surgery (0 day). At 14 and 21 days after ischemic surgery, blood perfusion of the ischemic hind limb had improved significantly in all treated groups compared with the vehicle group. In particular, although no significant differences in blood perfusion were detected between the CCL 150 and CCL 450 groups at 21 days after ischemic surgery, a high-dose of CCL (450 mg/kg/day) significantly ameliorated the signs of hind-limb ischemia earlier than a low-dose of CCL (150 mg/kg/day), at 14 days after surgical induction of ischemia.

To determine the angiogenic effects of CCL on microcirculation, we evaluated the capillary density in immunostained (anti-CD31) tissue from ischemic thighs and calf muscles. Positive structures were counted on five different microscopic fields per section under 200× magnification to determine capillary density (mean number of capillaries/mm²). Immunohistochemistry showed that the E2-treatment increased capillary density in ischemic muscles compared to the vehicle control. In addition, the capillary density significantly increased in both the low and high-dose CCL-treated groups (Fig. 2).

We determined whether CCL treatment promoted the expression of angiogenesis-related factors in serum and wound region tissue of ovariectomized and hind-limb ischemia-induced mice. The results revealed that the serum levels of the endothelin-1 and IGFBP-3 gene expression were significantly different between the treatment and vehicle groups. Moreover, the treatment with CCL increased endothelin-1 protein concentration in a dose-dependent manner (Fig. 3A). Next, we confirmed these findings using RT-PCR. The assessment of endothelin-1 and IGFBP-3 mRNA levels revealed a significant increase by 1.2- to 1.5-fold (Fig. 3B).

In parallel with protein array, we confirmed the effects of CCL on the mRNA expression of the angiogenesis-related marker genes VEGF, angiopoetin-1, and VEGFR2 in ischemic lesions of the hind limb. Tissue analyses revealed significant changes in the genes investigated (transcript-level) in response to CCL treatment (Fig. 4). Specifically, the expression of all three genes was upregulated in response to 150 and 450 mg/ml of CCL, indicating that CCL treatment was effective for wound healing and essential to accelerate angiogenesis.

We conducted the analyses of mouse inflammatory or pro-inflammatory cytokine expression to confirm the differences in the E2- and CCL-treated groups. The treatment with E2 and CCL led to secretion of high levels of CXCL12 (SDF-9) and CD54 (ICAM-1) compared to the vehicle groups (Fig. 5A). Moreover, mRNA expression of CXCL12 and CD54 was upregulated in the E2 and CCL groups (Fig. 5B). The results revealed that mRNA expression of the pro-inflammatory genes, TNF-α, IL-6 and IL-1β, was lower than that of the non-treatment group following the treatment with E2 and CCL (Fig. 6).

We evaluated the effects of CCL at 0, 1, 5, 10, 50, 100, 200 and 300 µg/ml on the viability of HUVECs. As shown in Fig. 7A, no significant cytotoxicity was observed in response to the treatment with 300 µg/ml CCL. Furthermore, the number of cells that migrated was significantly increased in VEGF and E2-treated groups, and this increase was also induced in a dose-dependent fashion by CCL treatment compared to the control (Fig. 7B). The tube formation increased in the groups treated with VEGF and E2 compared to the control groups, whereas tube organization was not induced by vinblastine. In CCL-treated groups, the induction of tube formation was observed at the dose of 100 µg/ml (Fig. 7C).