Functional effects of visfatin in isolated rat mesenteric small resistance arteries

Esra Akcabag a,*, Zeliha Bayram a, Ikbal Ozen Kucukcetin b, Gulbahar Uzun b, Sebahat Ozdem b, Sadi S. Ozdem a

Abstract

A new adipocytokine, visfatin is expressed in perivascular adipose tissue (PVAT) and exerts effects on vascular system in addition to its relationship with various pathological conditions. The present study aimed to investigate the functional effects of visfatin and the possible underlying mechanism(s) of the effects of visfatin in isolated rat mesenteric small resistance arteries. The study was conducted in small resistance arterial rings isolated from rat mesenteric vascular beds. While visfatin incubation did not produce significant alterations in contractile responses of mesenteric arterial rings to noradrenaline, relaxation responses to acetylcholine but not to sodium nitroprusside (SNP) were significantly reduced in endothelium-intact rings. The inhibitory effect of visfatin on responses to acetylcholine was not observed in endothelium-denuded preparations. Incubation of tissues with nicotinamide phosphoribosyl transferase (NAMPT) inhibitor FK866 or superoxide dismutase (SOD) reversed the inhibitory effects of visfatin on relaxation responses to acetylcholine. Co-incubation of visfatin with Nω-nitro-L- arginine methylester (L-NAME) did not produce a significant alteration in vascular responses to acetylcholine compared to L-NAME incubation alone. Mesenteric PVAT visfatin levels were significantly higher than and correlated positively with plasma visfatin levels. The results of our study indicated that visfatin-induced reductions in endothelium-dependent relaxations of rat isolated small resistance arteries are mediated by oxygen free radicals and a reduction in nitric oxide (NO) bioavailability. It was suggested that increment in systemic and/or local visfatin levels due to various pathologies including obesity and excessive weight gain may play a substantial role in initiation and/or propagation of vascular dysfunctions.

Keywords:
Adipocytokine
Nitric oxide
Perivascular adipose tissue
Small resistance arteries
Visfatin

1. Introduction

Adipose tissue acts as an active endocrine organ and produces a large number of bioactive factors called adipokines/adipocytokines. Adipocytokines can act locally in adipose tissue and reach different organs by systemic circulation, thus affecting the regulation of food intake-body weight, inflammation, reproduction, insulin sensitivity, immunity and vascular events (Guzik et al., 2006; Lau et al., 2005). An unbalanced increase in adipocytokines may also affect metabolic disorders such as obesity and type 2 diabetes mellitus, as well as insulin resistance, adipose tissue inflammation, chronic systemic inflammation and endothelial dysfunction (Karastergiou and Mohamed-Ali, 2010; Kumari and Yadav, 2018). In the development of metabolic diseases associated with cardiovascular complications, the role played by adipocytokines has recently become a curious research topic.
Visfatin, also known as pre-B-cell colony-enhancer factor and nicotinamide phosphoribosyl transferase (NAMPT), is a novel adipocyte- derived cytokine (adipocytokine) expressed ubiquitously in all tissues (Rongvaux et al., 2002; Samal et al., 1994). It plays a regulatory role in nicotinamide adenine dinucleotide (NAD) biosynthesis affecting NAD-dependent proteins such as sirtuins, poly (adenosine diphosphate-ribose) polymerases, mono (adenosine diphosphate-ribose) transferases, cluster of differentiation 38 and 157 through its enzymatic intrinsic activity and also has pro-inflammatory and immunomodulating properties through its cytokine-like extrinsic activity associated with many hormone-like signaling pathways and activation of some intracellular signaling cascades (for a detailed review, see Dakroub et al., 2020).
Visfatin is thought to be a proinflammatory adipocytokine associated with vascular damage and endothelial dysfunction (Liu et al., 2009; Romacho et al., 2009; Wang et al., 2009). It mediates arterial inflammation and endothelial dysfunction during early stages of obesity via an inflammasome dependent endothelial inflammatory response in mice (Xia et al., 2014). It also up-regulates markers of adipose tissue fibrosis in pre-adipocytes as well as obese children and adolescents suggesting that adipose tissue fibrosis due to visfatin-induced up-regulation in extracellular matrix proteins may be a possible link between visfatin and obesity-associated fibrosis and insulin resistance (Ezzati-Mobaser et al., 2020). Therefore, it has been associated with several cardiovascular-metabolic disorders (Dakroub et al., 2020). Furthermore, based on the findings of a meta-analysis, visfatin was proposed to be a promising biomarker for obesity, diabetes status, insulin resistance, metabolic syndrome and cardiovascular disease (Chang et al., 2011). On the other hand, despite increased circulatory levels implicating the role of visfatin in pathogenesis (Ezzati-Mobaser et al., 2020; Filippatos et al., 2010; Fukuhara et al., 2005; Hetta et al., 2018; Kabir et al., 2018; Sandeep et al., 2007), there are conflicting results regarding its expression and systemic levels in these cardiovascular-metabolic disorders. For example, both normal or decreased plasma visfatin levels were reported in atherosclerosis-related disorders (for a detailed review, see Filippatos et al., 2010) and gestational diabetes mellitus (Tsiotra et al., 2018). Similarly, although higher plasma visfatin levels were reported in obese people with visceral compared to subcutaneous fat deposits (Fukuhara et al., 2005; Kumari and Yadav, 2018), Berndt et al. found no difference in visfatin mRNA expression levels between subcutaneous and visceral fat accumulations (Berndt et al., 2005). Nevertheless, it was suggested that these controversial results do not rule out the possibility of an association between visfatin and these disorders, but rather suggest the existence of specific metabolic conditions dictating the plasma visfatin concentrations (Dakroub et al., 2020). Beside that apart from systemic circulatory levels, local perivascular adipose tissue (PVAT) levels may also be important in pathogenesis of certain cardiovascular–metabolic disorders; visfatin is released by a wide range of cell lines directly interacting with vascular cells (Stephens and Vidal-Puig, 2006) and locally synthesized visfatin may exert a paracrine effect on vascular tissue, as well. In accordance, experimental and human studies have shown that visfatin was expressed in PVAT of vessels such as aorta and coronary artery and has a variety of functions in the vascular system; visfatin produced in PVAT of the rat aorta stimulates cell proliferation in vascular smooth muscle cell cultures obtained from the same tissue via the extracellular signal-regulated kinase 1/2 and p38 signaling pathways (Wang et al., 2009). Furthermore, visfatin expression in periaortic and pericoronary fat tissues correlates positively with coronary atherosclerosis and increased visfatin expression in plaque rupture areas was reported in patients with coronary artery disease (Dahl et al., 2007; Spiroglou et al., 2010). Visfatin stimulates endothelial angiogenesis by increasing phosphoinositol 3 kinase/Akt and extracellular signal-regulated kinase 1/2 activation-induced increments in vascular endothelial growth factor and matrix metalloproteinases (Adya et al., 2008) and may also cause vascular endothelial inflammation by inducing adhesion molecules such as intracellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 via nuclear factor kappa-B-dependent mechanisms (Kim et al., 2007). Therefore, both the implication and the therapeutic target potentials of visfatin in cardiovascular–metabolic disorders need further investigations.
Although it is known that visfatin can also have direct effects on vascular reactivity, the number of studies investigating functional effects and the mediators involved in the functional effects of visfatin on vasculature is rather limited and the results are quite discordant; visfatin stimulated endothelial nitric oxide synthase (NOS) expression and activity in human umbilical vein and coronary endothelial cells (Lovren et al., 2009), and relaxed isolated rat aorta and mesenteric artery through endothelial NOS activation (Yamawaki et al., 2009). In contrary, flow-mediated dilation correlated negatively with plasma visfatin levels in type 2 diabetes mellitus (Hsu et al., 2016; Takebayashi et al., 2007) or chronic renal insufficiency (Yilmaz et al., 2008) and the decrease in circulating visfatin levels following renal transplantation was accompanied with an improvement in endothelial function assessed by flow-mediated dilation (Yilmaz et al., 2009). Furthermore, it was reported that visfatin abolished endothelium-dependent relaxations in human and rat mesenteric microvessels through a mechanism involving nicotinamide adenine dinucleotide phosphate (NADP) oxidase stimulation and relying on NAMPT enzymatic activity (Vallejo et al., 2011). These conflicting and limited result indicated that further studies are needed to clarify the effects of visfatin on vascular reactivity.
Therefore, in the present study, we investigated the functional effects and the mechanisms involved in functional effects of visfatin in isolated rat mesenteric small resistance arteries. Considering the systemic circulatory levels and the paracrine effect of locally produced visfatin, we also measured plasma and mesenteric PVAT visfatin levels in rats both to determine relations between systemic and local visfatin concentrations and to compare them with the concentrations inducing functional responses in mesenteric arterial rings.

2. Material and methods

2.1. Animals

This study was approved by the Akdeniz University Local Animal Ethics Committee (Protocol no: 2013.06.02) and was in accordance with the Declaration of Helsinki. Ten to twelve-weeks old male Wistar rats (210–400 g, n = 20) were used in the study. The animals were housed for a period of 12 weeks with free access to regular rat chow and water ad libitum.

2.2. Experiments in wire myograph

On the day of the experiment, rats were anesthetized by urethane (1 g/kg, i.p.) and after a midline abdominal incision, a portion of the small intestine was rapidly removed and placed in cold, well oxygenated (95% O2/5% CO2) Krebs–Henseleit solution (pH 7.4) of the following composition (mM): NaCl 118, KCl 5, NaHCO3 25, KH2PO4 1.0, MgSO4 1.2, CaCl2 2.5, and glucose 11.2. The animals were then sacrificed by cardiac incision. Third branches of the mesenteric arteries, approximately 150–200 μm in diameter, were dissected, cleaned of fat and connective tissues and cut into 1.8–2 mm-long segments under a dissecting microscope. Vessel rings were mounted in a Mulvany–Halpern wire myograph (Danish MyoTechnology, Aarhus, Denmark) with two tungsten wires (40 μm in diameter) inserted through the lumen of the vessel. After mounting the arteries, the wire myograph bath solution was changed to a fresh Krebs–Henseleit solution maintained at 37 ◦C and gassed continuously with 95% O2/5% CO2 (pH: 7.4). Subsequently, the arteries were rested for 10 min without tension prior to the normalization procedure. The internal diameter of each vessel was normalized as described by Mulvany and Halpern (1977). This was performed by stretching the vessel to a length that yielded a circumference equivalent to 90% of that given by an internal pressure of 100 mmHg. The isometric tension generated by the vessels (force tension divided by the length of the tissue, mN/mm) was recorded using isometric force transducers (Danish MyoTechnology, Aarhus, Denmark), connected to a computer-based data acquisition system (Biopac MP35, CA, USA). The preparations were allowed to equilibrate for 60 min before the start of the experiments. During this period, the bath solution was changed every 15 min.
Experiments were done on sets of two preparations, one containing and the other denuded of endothelium. After an equilibration period of 60 min, the presence of a functional endothelium was confirmed by the ability of acetylcholine (10− 6 M) to produce relaxation of tissues (>60%) precontracted with phenylephrine (10− 6 M). In order to denude the endothelium, intimal surfaces of mesenteric arterial rings were gently rubbed by insertion of human hair into lumen. Successful removal of the endothelium was confirmed by the inability of acetylcholine to induce relaxation in phenylephrine precontracted rings.
In the first part of the experiments, the effect of visfatin on the basal resting tone of the mesenteric arterial rings were investigated by adding cumulative concentrations of visfatin (1, 5, 15, 25, 35, 50 and 100 ng/ ml) into organ baths. In order to study the effect of visfatin on responses to noradrenalin, concentration-response curves to noradrenalin (10− 10 – 10− 5 M) were obtained in mesenteric arterial rings both in the absence and in the presence of visfatin incubations for 30 min at concentrations of 1, 5, 25, 50 and 100 ng/ml.
In the second part of the experiments, to study the effects of visfatin on endothelium-dependent and endothelium-independent relaxations, concentration-relaxation responses to acetylcholine (10− 10 – 10− 5 M) and sodium nitroprusside (SNP, 10− 10 – 10− 5 M), respectively were constituted in mesenteric arterial rings precontracted with submaximal concentrations of phenylephrine before and after 30 min of incubation of vessel preparations with 1, 5, 25, 50 and 100 ng/ml of visfatin.
Based on the findings obtained in preceding experiments, in the third part of the study, to investigate the role of nitric oxide (NO) and cyclooxygenase products in the effects of visfatin on relaxation responses in small resistance arteries from rat mesenteric beds, concentration-relaxation responses of phenylephrine precontracted vascular preparations to acetylcholine (10− 10 – 10− 5 M) were re- constituted after incubation of the tissues with the following agents for 20 min: cyclooxygenase inhibitor indomethacin (10− 5 M); blocker of the structural and inducible NOS isoforms Nω-nitro-L-arginine methylester (L-NAME); L-NAME (10− 4 M) and indomethacin (10− 5 M) combination; L-NAME (10− 4 M) and indomethacin (10− 5 M) and visfatin (100 ng/ml) combination. Furthermore, to test the specificity of the effect of visfatin, acetylcholine (10− 10 – 10− 5 M)-induced relaxation responses were obtained following incubation of tissues with visfatin (25, 50 and 100 ng/ml, 30 min) in the absence and presence of NAMPT inhibitor FK866 (10 μM, 30 min). The concentration of FK866 was selected on the basis of study by Vallejo et al. who reported that FK866 concentration-dependently (10 nM–10 μM) restored the impaired relaxation to acetylcholine (up to 3 μM) elicited by 50 ng/ml visfatin in rat microvessels, where the complete restoration was obtained with 10 μM (Vallejo et al., 2011).
In another set of experiments, the role of oxygen free radicals in the effect of visfatin on acetylcholine-induced vascular responses was investigated by obtaining the relaxation responses to acetylcholine in the presence of visfatin (15 ng/ml and 35 ng/ml) before and after incubation of mesenteric arterial rings with free oxidant radical scavenger superoxide dismutase (SOD, 100 U/ml) for 20 min.
The concentrations of all drugs were reported as the final molar concentrations in organ bath. Separate experiments were performed at intervals of at least 60 min. Each experimental group was performed in at least 6 mesenteric artery rings from different animals. 2.3. Measurement of plasma and mesenteric PVAT visfatin levels
Blood samples obtained from sacrificed animals on the day of experiment were taken into Lavender vacutainer tubes containing ethylene diamine tetra acetic acid. Following centrifugation of tubes within 30 min, plasmas were separated and stored at − 80 ◦C until the day of biochemical measurements. Mesenteric PVAT obtained by dissection were stored in liquid nitrogen until the day of measurement.
For protein extraction in mesenteric PVAT, frozen fat tissue samples were placed on ice and excision samples (200 mg) taken were dissolved in 200 μl PBS (0.01 mol/L; pH: 7.4) and homogenized in an ultrasonic homogenizer (30 s, 4 ◦C, pulse on 3 s, pulse off 5 s). Fat tissue homogenates were kept at − 80 ◦C for one night and melted on ice the next day. After two freeze-thaw cycles, homogenates were centrifuged at 15000 rpm for 30 min and the middle layer was used for the measurement. Visfatin levels in plasma and mesenteric PVAT were determined using a commercially available ELISA kit specific for rats (MyBioSource, Inc., San Diego, CA, USA, Catalog no: MBS4503321). Sensitivity of the kit was 0.55 ng/ml with a detection range of 1.56–100 ng/ml. Intra- and inter-assay coefficient of variations (CV) were <10% and <12%, respectively. Mesenteric PVAT levels were proportioned to tissue protein levels which were measured by the Lowry method (Lowry et al., 1951). PVAT visfatin levels proportioned to tissue protein levels were also corrected for body weights of the animals.

2.4. Drugs used in experiments

Visfatin, FK866, phenylephrine, acetylcholine, L-NAME, indomethacin, noradrenalin, SNP, SOD and salts for the Krebs-Henseleit solution were purchased from Sigma Chemical (St. Louis, Mo.). Visfatin, FK866, phenylephrine, acetylcholine, L-NAME, noradrenalin, SNP and SOD were dissolved in distilled water, indomethacin in ethanol. The final concentration of ethanol in the organ bath never surpassed 0.1%.

2.5. Statistical analysis

All values were expressed as mean ± SEM. Relaxation responses were expressed as percentages of the phenylephrine-induced contraction. The concentration of agonist which elicited a 50% maximal response (Emax) was designated as EC50, which was calculated by nonlinear regression. Sensitivity was expressed as pD2 (-Log EC50). Statistical analysis of the results was performed using Student’s t-test and one-way analysis of variance followed by Tukey post hoc test where appropriate. The relationship between plasma visfatin levels and visfatin measurements in PVAT was determined by Pearson Correlation Analysis. P values lower than 0.05 were considered significant.

3. Results

Neither visfatin nor NAMPT inhibitor FK866 caused significant changes in basal resting tones of vascular preparations from rats. Phenylephrine applied at submaximal concentration caused stable and sustained contractions in isolated rat mesenteric arterial rings (data not shown). Incubation of endothelium-intact mesenteric arterial rings with visfatin for 30 min at concentrations of 1, 5, 25, 50 or 100 ng/ml did not cause a significant alteration in contractile responses to noradrenalin (10− 10 – 10− 5 M) (Fig. 1). Visfatin caused significant alterations in relaxant responses to cumulatively applied concentrations of acetylcholine (10− 10 – 10− 5 M) only in endothelium-intact but not in endothelium-denuded small resistance arteries (Figs. 2 and 3). Based on these findings, further experiments were carried out with visfatin only in endothelium-intact preparations.
However, relaxation responses of phenylephrine precontracted mesenteric arterial rings to endothelium-independent relaxing agent SNP (10− 10 – 10− 5 M) were not altered significantly following incubations of vascular preparations with visfatin at concentrations of 1, 5, 25, 50 and 100 ng/ml for 30 min (Fig. 4, Table 1). Visfatin-induced inhibition in relaxation responses to acetylcholine was reversed almost completely by incubation of mesenteric arterial rings with NAMPT inhibitor FK866 (10 μM) for 30 min. Emax values for the acetylcholine-induced relaxations in the presence of visfatin at concentrations of 25, 50 and 100 ng/ml (37.22 ± 8.56%, 35.11 ± 8.98%, 34.66 ± 6.93%, respectively) increased significantly by the co- incubation of vascular preparations with FK866 (62.29 ± 8.16%, 61.05 ± 11.33%, 68.75 ± 13.45, respectively, p < 0.05 for all) and almost reached the control levels (74.55 ± 4.95%, 73.85 ± 6.15%, 73.53 ± 7.45%, respectively, p < 0.05 vs. visfatin alone, p > 0.05 for all vs. visfatin and FK866 co-incubations) (Fig. 5).
Incubation of endothelium-intact mesenteric arterial rings with L- NAME but not indomethacin resulted in significant reductions in relaxant responses to acetylcholine (Emax: 66.94 ± 6.55% vs. 21.00 ±1.22%, for control vs. L-NAME, respectively, p < 0.05). L-NAME- induced reductions in acetylcholine relaxations in endothelium-intact rat isolated mesenteric arteries did not increase further in the presence of indomethacin or visfatin at the highest concentration (100 ng/ml) (Fig. 6). Incubation of tissues with SOD (100 U/ml) for 30 min reversed the visfatin-induced reductions in relaxant responses of rat small resistance arteries to acetylcholine. Following SOD incubation, Emax value for acetylcholine in the presence of visfatin at 35 ng/ml (33.19 ± 6.62%) increased significantly (64.58 ± 5.2%) and was similar to control value (72.2 ± 4.26%).
Mean plasma and mesenteric PVAT visfatin levels of rats were 8.75 ± 0.81 ng/ml and 117.75 ± 12.47 ng/mg, respectively (n = 19). There was a significant positive correlation between plasma and mesenteric PVAT visfatin levels (r = 0.485, p = 0.035). Plasma visfatin levels also correlated positively with mesenteric PVAT visfatin levels corrected for body weight (r = 0.502, p = 0.034) (Fig. 8).

4. Discussion

In the present study, we have presented pharmacological evidence about both the functional effects of visfatin and the roles of NAMPT enzyme activity, endothelium, NO, cyclooxygenase products and oxygen free radicals in the effects of visfatin in small resistance arteries isolated from rat mesenteric vascular bed. To our knowledge, this is the first study in the literature investigating almost all possible mechanisms involved in vascular effect of the visfatin in the same study. In addition, by measuring plasma and mesenteric PVAT visfatin levels corrected for body weight in the same animal and examining the correlation between these parameters in relation with the observed functional vascular effects of a wide range of visfatin concentrations in the same study, we have provided new evidence that visfatin may be involved in vascular dysfunction in various pathologies with increased systemic and/or local visfatin levels.
The plasma concentration of visfatin under normal (physiological) or various pathological conditions in humans was reported to be in the range of 0.05 nM–0.25 nM (Hetta et al., 2018). Therefore, in the present study we used visfatin at concentrations covering both low, normal and high plasma concentrations in humans. We found that visfatin caused significant alterations neither in basal tonus nor in contractile responses of isolated rat mesenteric resistance arteries to endogenous vasoconstrictor noradrenalin even at very high concentrations used (Fig. 1), indicating that it does not have a direct role in physiological regulation of resistance arteries in rats. Consistent with this finding, Vallejo et al. reported that visfatin applied at concentrations of 50 and 100 ng/ml did not produce significant alterations in contractile responses to noradrenalin in rat mesenteric arteries (Vallejo et al., 2011). In contrary, contractile responses to noradrenalin was found to be significantly reduced in rat isolated thoracic aorta by 100 ng/ml visfatin incubation (Yamawaki et al., 2009) which suggested that there might be differences between conductance and resistance arteries in terms of the role of visfatin in regulation of contractile responses to endogenous vasoconstrictors.
In the present study, we have also investigated the effect of visfatin on endothelium-dependent and endothelium-independent relaxations in isolated resistance arteries from mesenteric vascular bed of rats. Visfatin incubation of phenylephrine precontracted rat mesenteric arterial rings with low, normal and high visfatin concentrations (1, 5, 25, 50 and 100 ng/ml) did not produce a significant alteration in responses to endothelium-independent vasorelaxant agent SNP. On the other hand, visfatin produced significant decreases in relaxation responses to endothelium-dependent agent acetylcholine at all concentrations used (1, 5, 25, 50 and 100 ng/ml). Decrement in maximal relaxations (Emax) was ranging concentration-dependently from 35% to 45% and accompanied by decrements in sensitivity (pD2) to acetylcholine reaching statistical significance only at the highest visfatin concentration used (Fig. 2, Table 1). These findings confirmed the results of the study by Vallejo et al. albeit small differences; in their study although visfatin pre- incubation, in agreement with our results, did not alter relaxant responses to SNP, relaxant responses to acetylcholine was impaired only by relatively high concentrations of visfatin (25, 50 and 100 ng/ml) but not with 10 ng/ml (Vallejo et al., 2011). Besides that, although acetylcholine efficacies (Emax) seemed similar at the highest visfatin concentration (100 ng/ml) in both studies, they seemed to be lower in their study at both 25 and 50 ng/ml visfatin concentrations compared to ours. Similarly, sensitivity to acetylcholine (pD2) particularly at higher visfatin concentrations (50 and 100 ng/ml) seemed to be lower than our corresponding findings. It is also worth noting that they did not studied the effect of lower visfatin concentrations (1 and 5 ng/ml) and presented the related sensitivity and efficacy data as figures not in numbers.
It is important to note that in our study the inhibitory effect of visfatin on endothelium-dependent relaxant responses was observed only in endothelium-intact but not in endothelium-denuded vascular rings. Both acetylcholine and SNP lead to NO-mediated soluble guanylate cyclase activation resulting in increased production of cyclic guanosine monophosphate (cGMP) and subsequent vascular smooth muscle relaxation. SNP acts as a direct NO donor whereas, acetylcholine leads to NO release from endothelial cells via NOS activation. Therefore, it might be suggested that the observed visfatin-induced reduction in endothelium-dependent relaxant responses of small resistance arteries in rats might be due to reduction in bioavailability of NO. In the present study, to determine the mechanisms that might play a role in the observed functional effect of visfatin in rat isolated resistance arteries, we obtained concentration-response curves to acetylcholine in the presence of blockers of different mediators known to be involved in endothelium-dependent relaxations of vascular tissues, alone and in combination with the highest concentration of visfatin (100 ng/ml). Incubation of the endothelium-intact arterial rings with the NOS blocker L-NAME resulted in a reduction in relaxant responses to acetylcholine by approximately 70%. On the other hand, cyclooxygenase inhibitor indomethacin neither produced a significant alteration in acetylcholine relaxations by itself, nor caused a further inhibition upon co-incubation in relaxant responses to acetylcholine observed with L-NAME. Taken together these findings indicated that relaxant effect of acetylcholine in rat mesenteric resistance arteries was mediated, to a great extent, by endothelium-derived NO but not by locally produced cyclooxygenase products. Furthermore, visfatin added at the highest concentration of 100 ng/ml to L-NAME and indomethacin co-incubation did not produce a further increment in maximal inhibitory effect obtained with L-NAME incubation, alone or in combination with indomethacin. More importantly, the maximal inhibition induced by the highest concentration of visfatin (100 ng/ml) on acetylcholine relaxations was markedly lower (~45%, Table 1) than those obtained with L-NAME, alone and in combination with highest visfatin concentration (~70% for both). Therefore, taken together all these finding supported the view that the inhibitory effect of visfatin on endothelium-dependent relaxations in isolated rat small resistance arteries are mediated by an interaction with the endothelium-derived NO. In support of this view, it has been shown that brachial artery flow-mediated dilation correlated negatively with plasma visfatin levels in type 2 diabetes mellitus (Takebayashi et al., 2007) and chronic renal failure (Yilmaz et al., 2008) and the decrease in circulating visfatin levels following renal transplantation was accompanied by an improvement in endothelial dysfunction assessed by flow-mediated dilation (Yilmaz et al., 2009). Furthermore, in accordance with our findings, Vallejo et al. showed a deterioration in endothelium-dependent but not in endothelium-independent relaxations by visfatin in human and rat mesenteric microvessels (Vallejo et al., 2011). Depending on their findings of prevention of relaxation impairment elicited by visfatin by NAMPT inhibitor FK866 and restored sensitivity to L-NAME in rat microvessels pre-incubated with visfatin and NADP oxidase inhibitor apocynin, they proposed that visfatin impairs endothelium-dependent relaxation mechanism involving NADP oxidase stimulation and relying on NAMPT enzymatic activity resulting in diminished NO bioavailability (Vallejo et al., 2011). In our study, in agreement with Vallejo et al., visfatin-induced inhibition in relaxation responses to acetylcholine was almost completely blocked by another NAMPT enzyme inhibitor FK866 (Fig. 5) which further supported the view that the inhibitory effect of visfatin on endothelium-dependent relaxations in small resistance arteries might be due to visfatin-induced reduction in bioavailability of NO (Vallejo et al., 2011). Contrastingly, it was reported that visfatin regulates endothelial NOS and Akt phosphorylation in endothelium-intact rat thoracic aortic rings to produce NO-mediated relaxation responses (Yamawaki et al., 2009), and stimulates endothelial NOS expression and activity in human umbilical vein and coronary endothelial cell cultures, leading to increased production of NO and cGMP formation (Lovren et al., 2009). Although the cause/causes of the differences between the results obtained in different studies regarding the vascular functional effects and the mechanisms involved in functional effects of visfatin are currently unknown and certainly need further research, it is known that variability in types and densities of pharmacological receptors, in ion transport mechanisms and in endothelial secretions depending on the size and location of the arteries may play an important role in heterogeneity in the responsiveness of vascular smooth muscles (Clark and Fuchs, 1997; Hwa et al., 1994; Mulvany and Aalkjaer, 1990).
It is known that superoxide anions scavenger enzyme SOD improves endothelium-dependent relaxation responses of resistance arteries in various pathological conditions (Diederich et al., 1994; Rodríguez-Manas et al., 1998˜ ). In the present study, SOD incubation restored the visfatin-induced impairment in endothelium-dependent relaxant responses of rat isolated mesenteric artery preparations (Fig. 7) suggesting that oxygen free radicals may be involved in inhibitory effect of visfatin on endothelium-dependent relaxations in isolated rat small resistance arteries. On the other hand, Vallejo et al. reported that co-incubation of rat mesenteric microvessels with SOD did not prevent the impaired relaxation to acetylcholine elicited by visfatin (Vallejo et al., 2011). Depending on the finding of prevention of visfatin-induced impairment by apocynin but not SOD, they proposed that NADP oxidase-induced intracellular release of superoxide anions may play a role in visfatin-induced impaired vasodilation. Although they have not presented their results with SOD incubation (data not shown), it might be suggested that superoxide anions may have a role in visfatin-induced impairment especially at high visfatin concentrations since reversal of acetylcholine relaxations by SOD to control levels in our study was observed only in vessels incubated with visfatin at 35 ng/ml, but not at 15 ng/ml concentrations.
Systemic visfatin concentration was reported as 15 ng/ml in healthy subjects (Chen et al., 2006; Lopez-Bermejo et al., 2006´ ) whereas much higher concentrations are expected in obese humans since, despite a few reporting the opposite, most studies showed that obesity is associated with increased circulating visfatin levels (Ezzati-Mobaser et al., 2020; Filippatos et al., 2010; Fukuhara et al., 2005; Goktas et al., 2013; Sandeep et al., 2007). Besides that, studies conducted in experimental animal models and humans have provided evidence that visfatin expression in PVAT is 3.7 and 1.8 times higher than those in subcutaneous and visceral adipose tissues, respectively (Spiroglou et al., 2010; Wang et al., 2009). Therefore, both circulating and local visfatin released from PVAT may exert effects on vascular tissues. In accordance, it was shown that changes in local PVAT visfatin levels may contribute to certain vascular pathologies (Dahl et al., 2007; Spiroglou et al., 2010). In the present study, we observed a significant impairment in endothelium-dependent vasorelaxation of small resistance arteries even at visfatin concentrations as low as 1 ng/ml and that seemed to be due to both oxygen free radicals and reduction in bioavailability of NO which is known to have vasoprotective effects against endothelial inflammation and thrombosis (Forstermann, 2010¨ ; Forstermann and Münzel, 2006¨ ). We also observed that mesenteric PVAT visfatin levels proportioned to tissue protein levels were substantially higher than plasma visfatin levels (117.75 ± 12.47 ng/mg and 8.75 ± 0.81 ng/ml, respectively) and there was a significant positive correlation between plasma and mesangial PVAT levels (r = 0.485, p = 0.035). Mesenteric PVAT visfatin levels corrected for body weight also correlated positively with plasma visfatin levels (r = 0.502, p = 0.034). Therefore, it seems possible that, apart from obesity, even excessive weight gain might cause an increment in systemic and local visfatin levels with resultant impairment in endothelial functions in resistance arteries.
Given that visfatin has marked pro-inflammatory as well as immunomodulating properties (Moschen et al., 2007), it is reasonable to expect that therapies aimed at reducing the effect of excess visfatin will improve the outcomes in diseases associated with elevated local and/or systemic visfatin levels. In accordance, it was shown that NAMPT inhibitors, such as FK866, has potent anti-inflammatory effects, and are novel compounds for therapy of various inflammatory diseases (Gehrke et al., 2014; Laiguillon et al., 2014; Matsuda et al., 2014; Montecucco et al., 2013; Nencioni et al., 2014; Nowell et al., 2012). In the present study, NAMPT inhibitor FK866 caused almost complete reversal of the visfatin-induced impairment in endothelium-dependent relaxations of mesenteric resistance arteries. Therefore, it might be suggested that the therapeutic potential FK866 in clinical usage may involve the opposition of the unfavorable effects of visfatin on endothelial functions in addition to its potent anti-inflammatory effects, particularly in diseases associated with elevated local and/or systemic visfatin levels.

5. Conclusions

Visfatin even at very low concentrations caused a significant reduction in endothelium-dependent relaxation responses of rat isolated small resistance arteries through oxygen free radicals and a reduction in NO bioavailability. Both mesenteric PVAT visfatin levels proportioned to tissue protein levels and corrected for body weight correlated positively with plasma visfatin levels in rats. Therefore, it was suggested that increment in systemic and/or local visfatin levels due to various pathologies including obesity and excessive weight gain may play a substantial role in initiation and/or propagation of vascular dysfunctions in these disorders.

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