Targeted Delivery of Salusin-α Into Rabbit Carotid Arterial Endothelium Using SonoVue

Preparation of Plasmid and SonoVue Mixture

Construction of pEGFP-Salusin-α and pcDNA3.1-Salusin-α.

Human salusin-α sequence (84-bp), synthesized by Shanghai Biotechnology, was inserted between the XhoI and BamHI digestion sites, into pEGFP-N1 vector (Invitrogen Life Technologies, Carlsbad, CA, USA) to generate pEGFP-salusin-α.¹⁷ Similarly, the salusin-α sequence was cloned into pcDNA3.1 (Invitrogen Life Technologies) using the same two digestion sites and pcDNA3.1-Salusin-α was generated. The polymerase chain reaction (PCR) forward primer sequence was.

5′-TGTGGGATCCGGTGCCCTTCCTCCCGCT-3′ and the PCR reverse primer sequence was 5′-TATACTCGAGTCCCTTGGCTCCAGGCCC-3′.

The two plasmids were sequenced to verify the success of molecular cloning. High-purity plasmids were prepared on a large scale for use in the experiments described below (Invitrogen DNA Purification Kit. No. 12280096A).

Based on our previous study,¹⁷ we prepared the mixture in the ratio of 20 μL SonoVue (45 μg/mL) solution and 1 μL plasmid (1 μg/μL) solution and tested the adhesion rates of different plasmids and SonoVue, as described below.

SonoVue Adherence Ratio to Plasmids

To evaluate the adherence ratio of SonoVue, varying amounts of SonoVue (45 mg/mL), which were prepared according to the manufacturer's instructions, were mixed with the same amount of plasmids (1 mg/mL). The total volume was the same after 0.9% sodium chloride (NaCl) solution was added to each mixture. The mixture was incubated for 15 minutes at room temperature and separated into two layers. The upper layer had a very thin white layer, which contained SonoVue adhered to plasmid molecules. The lower layer was a thick solution including free plasmids not included in SonoVue microbubbles. After incubation, ultraviolet spectrophotometry was performed to test plasmid concentration in the lower layer; absorbance (A) of each mixture was determined by measuring the optical density at 260 nm (A₂₆₀) and 280 nm (A₂₈₀) and using the following equation:

(from the Cold Spring Harbor online protocol, the plasmid concentration and adherence ratio were calculated)

Rabbit Grouping and Plasmid Transfection

All experimental New Zealand white rabbits (~2.5 kg) were obtained from Hubei Medical Experimental Animal Center and treated following protocols approved by the Institute of Animal Care and Use Committee of Hubei University of Chinese Medicine (approval document number 2015025, time: 2015-6-3). As described in the Guide and the Animal Welfare Act Regulations, rabbits were maintained in our laboratory under standard conditions and fed an ordinary diet every day.^{17, 18}

In order to analyze the transfection efficiency of pEGFP-salusin-α and pcDNA3.1-Salusin-α into the arterial tunica intima, nine rabbits were divided into three groups: the pEGFP-salusin-α (pES) transfection group (n = 3), for which successful transfection of vascular endothelium was judged by expression of the enhanced green fluorescent protein (EGFP) gene; the pcDNA3.1-Salusin-α (pcS) transfection group (n = 3) where transfection efficiency was measured by checking the expression of salusin-α; and the pH 7.4, phosphate-buffered saline (pBS) group (n = 3) as the control group, where only a mixture of phosphate-buffered saline (PBS) pH 7.4 (containing no plasmid) and SonoVue was administered.

The plasmid transfection method was the same as that used in our original study.^{17, 18} In brief, a mixture of 100 μL of plasmids (1 μg/μL) and 2 mL of SonoVue (45 μg/mL) was incubated for 20 minutes at room temperature’ and then mixed and immediately injected through a 22-gauge intravenous cannula into the ear vein of anesthetized (pentobarbital sodium 25 mg/kg; Invitrogen) rabbits. After injection, the right common carotid artery was ultrasonicated for 3 minutes until SonoVue microbubbles had been largely disrupted from the circulatory system. This transfection method is based on the hypothesis of transport mechanism of plasmid into vascular endothelium by SonoVue, shown in Figure 1. During the course of the experiment, plasmid transfection was carried out after 2 weeks with one transfection cycle being performed each week, once a week. Two weeks later, all rabbits' blood and left and right common carotid arteries were obtained. To minimize pain, rabbits were anesthetized and prevented from observing the culling process.

Baseline and Contrast-Enhanced Ultrasonography Examination Settings

All operating procedures were the same as described in our previous studies.^{17, 18} Table 1 shows the baseline and contrast-enhanced ultrasonography examination settings of the clinical ultrasound scanner (Siemens Acuson Sequoia 512, Siemens, Germany) used in this study. The conventional carotid artery examination mode was used for baseline ultrasound examination, and the mechanical index (MI) was 1.5. Low MI real-time contrast-enhanced ultrasonography (CEUS) examination was carried out with contrast pulse-sequencing imaging mode, MI was 0.18. Clinical ultrasound scanner detection data were stored on a hard disk.

Rabbit Blood Parameters and Vascular Pathology

Blood was collected from all rabbits at the end of experiments and EDTA-2K was used as anticoagulant. The following blood parameters were measured according to the laboratory blood test protocol: white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HB), hematocrit (HCT), platelet count (PLT), total protein (TP), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), creatinine (CREA), and glucose (GLU). Blood test values were compared between pBS and pcS groups.

To investigate vascular intimal hyperplasia, histopathological examination of vascular tissue sections was performed. Similar to the routine pathological examination previously described,^{17, 18} paraffin-embedded blocks of carotid arterial tissue obtained from rabbits in each group were sliced into 4-μm thick sections, stained with hematoxylin–eosin (HE), and examined by a trained pathologist. Using an Axiophot microscope (Zeiss, Austin, TX, USA) and a digital camera (Leaf Systems Lumina, Southborough, MA, USA), magnified images were captured and processed using a software from Optima Imaging Analysis Systems (Version 6.5, Media Cybernetics, Silver Springs, MD, USA).

Arterial sections from the animals in the pES group were harvested after 2 weeks with one transfection cycle being performed each week, once a week. Fast-frozen sections were prepared for checking EGFP expression with fluorescence microscopy, and for testing salusin-α level in arterial tissue by western blotting.

Western Blot and ELISA

In order to analyze protein expression in the carotid arteries of the rabbits, western blotting was performed as described in our previous studies.^{17, 18} Carotid artery samples were homogenized in 1 mL lysis buffer (50 mM NaCl, 10 mM Tris, 1 mM EDTA, 1 mM PMSF, 0.5 mM Na₃VO₄·12H₂O, 50 mM NaF, and 1 mM benzamidine), and centrifuged at 12,500g for 15 minutes at 4°C. Protein concentration was determined using BCA Protein Assay Reagents (Pierce Com., Rockford, IL, USA). For each sample, equal amounts of protein lysate (20 μg) were loaded onto 7.5% sodium dodecyl sulfate-polyacrylamide gels for electrophoresis and then transferred onto polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The membranes were blocked in PBS supplemented with 3% (w/v) bovine serum albumin for 1 hour at room temperature. The membranes were cut into strips according to protein molecular weight standard, and the strips expected to contain different molecular weight proteins were incubated overnight at 4°C with different primary antibodies, as follows: anti-EGFP rabbit polyclonal antibodies (OpenBiosystems, Ohio, USA; cat. no. CAB4211), anti-salusin-α rabbit polyclonal antibodies (Yansheng, Shanghai, China; cat. no. YS-KT2539), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Santa Cruz, CA, USA; cat. no. sc-25778). Horseradish peroxidase-conjugated goat anti-rabbit IgG2a antibody (Santa Cruz; cat. no. sc-2061) was used as a secondary antibody. Western blots were visualized using enhanced chemiluminescence detection reagents (Sigma, St. Louis, MO, USA) following manufacturer's instructions. Protein bands were quantified by scanning with Bio-Rad GelDoc XR and Chemi Doc XRS systems (Bio-Rad, Hercules, CA, USA), and analyzed using Quantity One 1-D Analysis Software Version 4.6.3 (Bio-Rad).

Human salusin-α ELISA kits were obtained from USCN Life Science Inc. (Wuhan, China). Salusin-α standard curves were generated according to manufacturer's instructions. Before examining the samples, the best dilution of serum samples was determined. A microtiter plate provided with the kit was pre-coated with a salusin-α antibody. Standards or samples were added to the appropriate microtiter plate wells with a biotin-conjugated salusin-α polyclonal antibody and avidin-conjugated to horseradish peroxidase (HRP), and incubated at 37°C. TMB (3, 3′, 5, 5′ tetramethyl-benzidine) substrate solution was added to each well, and the plates were incubated at 37°C until the enzyme-substrate mixed solution exhibited a change in color. The enzyme-substrate reaction was terminated by the addition of a sulfuric acid solution, and the color change was measured spectrophotometrically at a wavelength of 450 nm. The concentration of salusin-α in the samples was detected by comparing the OD values of the samples with the standard curve depending on the dilution.

Statistical Analysis

All data are presented as mean ± standard deviation. SPSS (version 19.0; SPSS, Chicago, IL, USA) was used for statistical analysis, and the mean results of different groups were analyzed by ANOVA to determine statistical significance. Differences with P values of less than .05 were considered significant. All experiments were performed three times.

Adhesion of Plasmid to SonoVue

The nucleotide sequence encoding for the vasoactive peptide salusin-α was synthesized by Shanghai Sangon, and is shown in Figure 2A. Salusin-α sequence was amplified by PCR and cloned into pEGFP-N1, to obtain pEGFP-salusin-α. pEGFP-salusin-α was then sequenced and its sequence was analyzed using Addgene website sequence analysis software to verify correct cloning (Figure 2B). The same was done for plasmid construct pcDNA3.1-Salusin-α.

As shown in Figure 3, we performed a comparative analysis of SonoVue adhesion rates to the four plasmids: pEGFP-N1, pEGFP-salusin-α, pcDNA3.1-Salusin-α, and pcDNA3.1. There was no difference among SonoVue adhesion rates to the four plasmids, suggesting that the sequence cloned into the plasmid did not affect SonoVue adhesion rate to the plasmid. SonoVue adhesion rate to all plasmids was 89%. In the present study, we chose pEGFP-salusin-α and pcDNA3.1 Salusin-α plasmids for transfection into the vascular endothelium.

Analysis of Rabbit Carotid Arterial Endothelium after Transfection with pEGFP-Salusin-α

The common carotid artery and its concomitant nerve were surgically dissected from anesthetized rabbits, which had been previously transfected with pEGFP-salusin-α (Figure 4A). The common carotid artery segments that had been transfected with plasmids were obtained (Figure 4B). To check whether targeted transfection had been successful at transferring the plasmid only locally where ultrasonication had been applied, the pES group was divided into two subgroups: pES-R for the right common carotid artery group and pES-L for the left common carotid artery group, because the left common carotid artery had not been subjected to ultrasonication and we were not expecting to see any EGFP expression there; while the right common carotid artery had been ultrasonicated with the SonoVue plus plasmid mixture, we were expecting to see EGFP expression there. As shown in Figure 4E for the pES-R subgroup, we achieved successful transfection of pEGFP-salusin-α in endothelial cells of the right common carotid artery endothelium. In fact, the right common carotid arterial endothelium showed green fluorescence, in contrast to the left common carotid arterial endothelium of the pES-L subgroup shown in Figure 4F and the common carotid arterial endothelium of the rabbits in the pBS group (Figure 4G), which did not show any EGFP expression. Both histological sections of common carotid artery segments, which had been transfected with pEGFP-salusin-α plasmid (Figure 4C), and histological sections of common carotid artery segments dissected from rabbits belonging to control groups (pES-L and pBS), which had not been transfected with the plasmid (Figure 4D), did not show any pathological changes. Gene expression of the transfected plasmid was further detected by western blotting. EGFP gene expression was apparent only in the right common carotid artery endothelium in the pES-R subgroup (Figure 4H, top). Salusin-α was expressed in all groups (Figure 4H, middle), which was consistent with Shichiri et al's report that Salusin-α was widely expressed in various tissues.¹⁹ Figure 4I shows that Salusin-α expression was greater in the pES-R group than in the other groups, and no EGFP protein expression was observed in the pES-L and pBS groups. These data confirm that the target salusin-α sequence was efficiently and site-specifically transfected into vascular endothelial cells, and we observed that its expression was consistent with the results of our previous studies.¹⁸

Evaluation of pcDNA3.1 Salusin-α Transfection of Rabbit Carotid Arterial Endothelium

As for pEGFP-salusin-α transfection, also for the transfection of plasmid pCDNA3.1-Salusin-α, a control group indicated as pBS group, which was treated with only PBS not containing the plasmid, and an experimental group, which was treated with the pcDNA3.1-Salusin-α plasmid indicated as pcS group, were set up. The expression plasmid pcDNA3.1-Salusin-α was transfected into the rabbit carotid artery endothelium, and the expression of salusin-α in each group was analyzed. To analyze the site-specific transfection of pcDNA3.1-Salusin-α, the pcS group was divided into two subgroups: pcS-R and pcS-L, representing the right (pcS-R) and the left (pcS-L) common carotid artery endothelial tissue. The expression of the salusin-α peptide in the pcS-R group was obviously higher than that in the pcS-L and pBS groups, but there was no significant difference in salusin-α expression between the pcS-L and the pBS group (Figure 5A). This confirmed targeted transfection of pcDNA3.1-Salusin-α plasmid into the carotid arterial endothelium and showed that the transfected sequence coding for the salusin-α peptide was expressed correctly.

We performed ELISA to detect plasma salusin-α levels in pBS and pcS groups (Figure 5B) and found that salusin-α values were 20.65 ± 2.38 pmol/L for the pBS group and 21.15 ± 2.44 pmol/L for the pcS group, which were not statistically different (P = .82). Therefore, transfection of pcDNA3.1-Salusin-α into the common carotid arterial endothelium does not seem to affect the plasmatic concentration of salusin-α. This could be due to the fact that the transfected and overexpressed salusin-α is not secreted into the blood stream and is retained inside the endothelial cells. This aspect warrants further studies for clarification.

Evaluation of Rabbit Blood Parameters

Before sacrificing the rabbits, we looked at the differences in various blood parameters of rabbits in the pBS and pcS groups (Table 2). The parameters included were routine blood test parameters such as blood cell counts, liver function, myocardial enzymes, renal function, and blood glucose, and there was no difference in any of the parameters between the two groups (P > .05). This indicated that transfection of the plasmid into the rabbit common carotid artery endothelium had no effect on these parameters.