Am J Physiol Cell Physiol 306: C123–C131, 2014. First published November 20, 2013; doi:10.1152/ajpcell.00164.2013.

CALL FOR PAPERS

Stem Cell Physiology and Pathophysiology

Bone marrow mononuclear cell angiogenic competency is suppressed by a high-salt diet Jamie R. Karcher1,2 and Andrew S. Greene1,2 1

Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin; and 2Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin

Submitted 3 June 2013; accepted in final form 14 November 2013

Karcher JR, Greene AS. Bone marrow mononuclear cell angiogenic competency is suppressed by a high-salt diet. Am J Physiol Cell Physiol 306: C123–C131, 2014. First published November 20, 2013; doi:10.1152/ajpcell.00164.2013.—Autologous bone marrow-derived mononuclear cell (BM-MNC) transplantation is a potential therapy for inducing revascularization in ischemic tissues providing the underlying disease process had not negatively affected BM-MNC function. Previously, we have shown that skeletal muscle angiogenesis induced by electrical stimulation is impaired by a high-salt diet (HSD; 4% NaCl) in Sprague-Dawley (SD) rats. In this study we tested the hypothesis that BM-MNC angiogenic function is impaired by an elevated dietary sodium intake. Following 1 wk on HSD, either vehicle or BM-MNCs derived from SD donor rats on HSD or normal salt diet (NSD; 0.4% NaCl) were injected into male SD rats undergoing hindlimb stimulation. Administration of BM-MNCs (intramuscular or intravenous) from NSD donors, but not HSD donors, restored the angiogenic response in HSD recipients. Angiotensin II (3 ng·kg⫺1·min⫺1) infusion of HSD donor rats restored angiogenic capacity of BM-MNCs, and treatment of NSD donor rats with losartan, an angiotensin II receptor-1 antagonist, inhibited BM-MNC angiogenic competency. HSD BM-MNCs and NSD losartan BMMNCs exhibited increased apoptosis in vitro following an acute 6-h hypoxic stimulus. HSD BM-MNCs also had increased apoptosis following injection into skeletal muscle. This study suggests that BM-MNC transplantation can restore skeletal muscle angiogenesis and that HSD impairs the angiogenic competency of BM-MNCs due to suppression of the renin-angiotensin system causing increased apoptosis. high-salt diet; bone marrow mononuclear cells; angiogenesis; reninangiotensin system ANGIOGENESIS IS ESSENTIAL for a variety of physiological processes including wound healing and development, as well as in pathological conditions including tumor growth (18) and ulcer formation (34). Insufficient angiogenesis can cause tissue ischemia or a restriction in blood supply with possible damage or dysfunction of tissue. Ischemia is common to several cardiovascular diseases including heart disease, transient ischemic attacks, and cerebrovascular strokes (51). Therefore, interventions that promote revascularization of ischemic tissue offer an important therapeutic approach because they can protect cells from prolonged ischemia and death and in some cases may help repair damaged tissue. Despite many advances in treatments for patients with ischemic-related diseases, most current clinical

Address for reprint requests and other correspondence: A. S. Greene, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (e-mail: [email protected]). http://www.ajpcell.org

treatments do not produce sustained revascularization of ischemic tissue. Increased dietary salt intake can inhibit blood vessel growth (43, 44) that in turn causes vascular rarefaction (3, 8, 23), rapid fatigue of muscle (37, 46), and an impaired training effect (43, 44). In the Sprague-Dawley (SD) rat, increased dietary salt does not induce hypertension but does induces microvascular rarefaction (22) and limits microvessel growth (44). The hypertension-independent effects of high salt on microvascular structure and function have been largely attributed to the suppression of renin-angiotensin system (RAS) by inhibition of renal renin release. The RAS has been shown to be a pivotal player in angiogenesis (1, 2) with angiotensin II (ANG II), the main effector peptide of the renin-angiotensin system, having important angiogenic properties. The formation of new blood vessels is initiated by signals that cause the stimulation of endothelial cells (ECs); degradation and invasion of the basement membrane, migration, and proliferation of ECs; and finally, the formation of new capillaries by lumen formation and basement membrane deposition (7, 17). Several mechanisms of RAS involvement in creating a permissive environment for angiogenesis have been proposed, including stimulation of EC proliferation (52) and increase in expression of specific growth factors, including vascular endothelial growth factor (VEGF) and its receptors (41). Recent studies have focused on the use of adult stem cells or progenitor cells, especially bone marrow-derived stem cells, as a therapeutic option for cardiovascular diseases. Bone marrow mononuclear cells (BM-MNCs) have been shown to increase blood vessel growth and either stop or reverse the progression of diseases including pressure ulcers in humans (48), chronic ischemia heart disease in pigs (50), pulmonary hypertension in dogs (32), and type 2 diabetes (55). BM-MNCs are also capable of increasing angiogenesis with critical limb ischemia in humans (54). However, several studies have now shown that the effectiveness of BM-MNC transplantation can depend on the health of the donor with demonstration of BM-MNC dysfunction observed in cardiovascular disease (57), heart failure (26), and type 2 diabetes (16, 58). The present study takes advantage of a normotensive rat model to determine the pressure-independent effects of dietary salt intake on bone marrow function. We have previously shown that BM-MNC function can be altered by both the genetic background and the disease state of the donor rat (11) such that normal function may be dependent on an active RAS. For effective autologous cell therapies, it is crucial to understand how adult stem cell function is impaired in disease and

0363-6143/14 Copyright © 2014 the American Physiological Society

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HIGH SALT INHIBITS BONE MARROW FUNCTION

whether impaired stem cells can be modified to enhance their therapeutic effectiveness. In the current study we tested the hypothesis that HSD through the suppression of the RAS would inhibit BM-MNC function and that this impairment would result in failure of transplant to restore angiogenesis. METHODS

Animal care and electrical stimulation surgery. All animal protocols were approved by the Medical College of Wisconsin (MCW) Institutional Animal Care and Use Committee. Animals were housed and cared for in the MCW Animal Resource Center and were given food and water ad libitum. In this study we describe two groups of SD rats: donors and recipients. BM-MNCs were harvested from donor rats and recipient rats received BM-MNC or vehicle transplantation along with electrical stimulation. One tibialis anterior (TA) muscle of the recipient rats was electrically stimulated for 8 h per day for 7 consecutive days as previously described (29). Briefly, male SD rats, 7 to 9 wk old, were anesthetized with an intramuscular injection of a mixture of ketamine (100 mg/kg), xylazine (50 mg/kg), and acepromazine (2 mg/kg). Under aseptic conditions, a miniature battery-powered stimulator that was previously designed and validated for chronic studies by our laboratory (29) was implanted and secured in place. A pair of electrodes were secured to the muscle of the right hindlimb in close proximity to the common peroneal nerve (29). The rats were allowed to recover before the initiation of the stimulation period the following day. Analysis of vessel density. The TA muscles from both hindlimbs of electrically stimulated rats were harvested and fixed in 0.25% formalin for 24 h. Each muscle was sliced on a sliding microtome to a thickness of ⬃75 ␮m. The muscle slices were stained in a 30 ␮g/ml rhodamine-labeled Griffonia simplicifolia I lectin (Sigma) for 3 h on a rocker. The samples were rinsed several times and then mounted on microscope slides as previously described (21). The sections were visualized with a video fluorescent microscope system (Nikon E-80i microscope with Q-Imaging QIClick camera, ⫻200, microscope field ⫽ 0.155 mm2) with fluorescent light (excitation 555 nm and emission wavelength 580 nm). Thirty to forty representative fields from each muscle were digitally photographed and computer analyzed by Metamorph Image Quant software (Molecular Devices) as previously described (45). Microvessel density was expressed as the mean number of vessel-grid intersections per microscope field. BM-MNC isolation. SD donor rats were placed on a normal-salt diet (NSD; 0.4% NaCl Dyets) or a high-salt diet (HSD; 4% NaCl Dyets) for 7 days before cell isolation. Donor rats were killed with an overdose of beuthanasia, and their BM-MNCs were harvested as previously described (14, 42). Briefly, bone marrow was harvested from the tibias and femurs by flushing with MCDB131 (E3000-01B; US Biologicals) with 5% heat-inactivated fetal bovine serum (FBS;

A

B G1

SSC-A

Fig. 1. Bone marrow mononuclear cell (BMMNC) composition as determined by flow cytometry analysis. A: density plot of forward scatter (FSC) vs. side scatter (SSC) of BM-MNCs showing gates for granulocytes (G1), monocytes (G2), and lymphocytes (G3). B: graphical representation of flow cytometry results of the BM-MNC composition. Only the groups that underwent a surgical procedure [high-salt diet (HSD) ⫹ ANG II and HSD ⫹ saline] had altered BM-MNC composition. S, group that underwent surgery. *Significant difference vs. NSD. #Significantly different vs. HSD (P ⱕ 0.05; n ⫽ 4). *Significant difference vs. normal-salt diet (NSD). #Significantly different vs. HSD. (P ⱕ 0.05; n ⫽ 4).

GIBCO by Invitrogen). The mononuclear cells were then isolated from the bone marrow by passing the cells through Histopaque (Sigma, 10831) by centrifugation. The mononuclear cells were isolated, washed, and then resuspended in MCDB131 media containing 5% heat-inactivated FBS. Cell number and viability was determined using the Countess automated cell counter (Life Technologies). Cells were washed free of media and diluted appropriately for injection. BM-MNC composition. The composition of the bone marrow was determined by light scatter analysis by FACS as previously described (5, 6). Following harvest of cells from donor rats, the cells were fixed with 2% paraformaldehyde and stained with 4=,6-diamidino-2-phenylindole dihydrochloride (DAPI; D8417; Sigma-Aldrich) for 30 min at room temperature to exclude debris and dead cells. A minimum of 1,000,000 events were recorded from each sample on a LSRII flow cytometer (BD Biosciences). Lymphocyte, monocytes, and granular sized cells were determined based on their size and cell complexity or granularity (Fig. 1A). BM-MNC transplantation. Recipient SD rats on a HSD (4% NaCl) or NSD (0.4% NaCl) were either injected intramuscularly into the stimulated TA muscle or intravenously into the tail vein. Recipients received 6 ⫻ 106 BM-MNCs in 0.2 ml of Dulbecco’s phosphatebuffered saline without calcium or magnesium (DPBS) or 0.2 ml DPBS alone (4 separate injection sites with ⬃50 ␮l per injection) as previously described (14). The unstimulated limb did not receive direct intramuscular injections. The recipient rats were divided into three groups: 1) SD rats on a NSD or HSD that received 0.2 ml of DPBS injection, 2) SD rats on HSD that received 6 ⫻ 106 cells BM-MNCs from SD rat on a NSD, and 3) SD rats on HSD that received 6 ⫻ 106 cells BM-MNCs from SD rat on a HSD. Immediately after the cell or DPBS injections, a battery-powered stimulator was implanted as described above. After 7 days of electrical stimulation the rats were euthanized and tissues were collected for analysis. In vivo ANG II infusion and oral losartan treatment in donor rats. Additional BM-MNC donor SD rats on a HSD received either saline or saline with ANG II (A9525; Sigma) at a subpressor dose of 3 ng·kg⫺1·min⫺1 intravenously for 7 days before BM-MNC harvest as described previously (44). Recipient rats, or rats that receive transplantation of BM-MNCs and electrical stimulation, were not treated with ANG II. Briefly, SD rats were anesthetized with intramuscular injection of a mixture of ketamine (100 mg/kg), xylazine (50 mg/kg), and acepromazine (2 mg/kg) and a catheter was securely inserted into the right jugular vein. Conscious rats were then connected to a multisyringe pump to provide a continuous intravascular infusion of saline alone or saline containing ANG II. After 7 days, BM-MNCs were harvested and resuspended in DPBS for implantation as described above. An additional group of SD donor rats on a NSD were given losartan (50 mg/day; Merck) orally by adding it to the rat’s normal drinking water for 5 days before BM-MNC isolation (1, 2). Recipient rats did not receive losartan treatment.

G2

G3

% cells in total BM-MNC

C124

100 80

*#

60 40 20 5 4 3 2 1 0

FSC-A

AJP-Cell Physiol • doi:10.1152/ajpcell.00164.2013 • www.ajpcell.org

*

*#

*#

*

*#

Lymphocytes Monocytes

Granulocytes

C125

HIGH SALT INHIBITS BONE MARROW FUNCTION

Immunoblot to determine cleaved caspase-3 protein level. BMMNCs from SD rats on a NSD, HSD, HSD with saline infusion, HSD with ANG II infusion, or NSD with losartan treatment were harvested as described above. Cells were placed in normoxic (21% O2, 5% CO2) or hypoxic conditions (2% O2, 5% CO2) at 37°C for 6 h and were flash frozen in mammalian protein extraction reagent (MPER; Thermo Fisher Scientific) containing protease inhibitors (Roche). Cells were sonicated and centrifuged at 3,000 g for 5 min. The amount of protein in each sample was determined using the Micro BCA Protein Assay Kit (Thermo Fischer Scientific). Protein was loaded on a 15% polyacrylamide gel and then blotted onto a nitrocellulose membrane. Membranes were incubated with a rabbit polyclonal antibody for cleaved caspase-3 [anti-cleaved caspase 3, Cell Signaling no. 9664L, dilution 1:900 in 2% nonfat dry milk (NFDM)] overnight at 4°C and after serial washes, with the secondary antibody [(H ⫹ L)-horseradish peroxidase-conjugated goat anti-rabbit, Bio-Rad no. 170 – 6515, dilution 1:5000 in 2% NFDM] for 2 h at room temperature. Immunoblots were visualized by chemiluminescence (Pierce), followed by autoradiography. The 17- and 19-kDa protein bands were quantified. Membranes were stained with Ponceau S (Sigma) to confirm equal protein loading. A population of BM-MNCs exposed to ultraviolet light was used as positive control. Results were normalized by Ponceau S and positive control expression. The results were expressed as relative densitometric values using the Metamorph Image Quant software (Molecular Devices). In vivo BM-MNC apoptosis. BM-MNCs were harvested as described above. Cells were labeled with quantum dots (Qdot from Invitrogen Qtracker 625 cell labeling kit, A10198). Two microliters of component A and B were mixed with 1 ⫻ 107 cells for 45 min at 37°C. Labeled cells were thoroughly washed before transplantation. Then, 1 ⫻ 106 Qtracker labeled BM-MNCs were transplanted via intramuscular injection into both the unstimulated and stimulated limb of HSD SD recipient rats undergoing electrical stimulator implantation as described above. Electrical stimulators were turned on for one round of 8 h of stimulation. The recipients were euthanized, and tissues were collected for analysis. The TA muscles of both unstimulated and stimulated limbs were fixed in 0.25% formalin for 24 h. The muscles were sliced as described above. Muscle slices were stained with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) from Promega per manufacturer’s recommendations. Immunofluorescence microscopy was performed with an ⫻40 objective. BM-MNCs were identified by red fluorescence (Qdots), and

apoptotic cells were determined by green fluorescence (TUNEL). Apoptotic BM-MNCs were detected by colocalized red and green fluorescence. Quantification of apoptotic BM-MNCs was expressed as percentage of Qdot positive and TUNEL positive BM-MNCs over nonapoptotic BM-MNCs (Qdot positive, TUNEL negative) per image field. Quantitative PCR to detect HIF-1␣ and VEGF. BM-MNCs were harvested as described above. Cells were incubated for 6 h at normoxic (21% O2, 5% CO2) or hypoxic conditions (2% O2,5% CO2) at 37°C and total RNA was purified using Qiagen RNeasy Mini Kit per manufacturer’s instructions. Purified RNA was quantified and tested for quality by using the Aglient RNA 6000 Nano Kit and run on a 7900HT real-time PCR machine (Applied Biosystems). Samples were run with the Taqman one-step kit (Applied Biosystems) per manufacturer’s instructions and the following oligos: hypoxia inducible factor-1␣ (HIF-1␣) forward 5-GTTCACAAATCAGCACCAAGC and HIF-1␣ reverse 5-GAACATCAAGTCAGCAACGTG; VEGF-A forward 5-TCTGCGGATCTTGGACAAAC and VEGF-A reverse 5-CAGATGTGAATGCACACCAAAG primers. Real-time PCR analysis was performed as described by Knoll et. al (27). HIF-1␣ and VEGF-A mRNA expression was normalized to endogenous 18S rRNA. Statistical analysis. Muscle and body weights were evaluated using independent paired t-tests. To evaluate the significance of differences in vessel density between stimulated and unstimulated sides of the TA muscle, a paired t-test (unstimulated vs. stimulated) and one-way ANOVA were performed. To evaluate the significance of differences in cleaved caspase-3 expression, and one-way ANOVA was performed. A significance value of P ⱕ 0.05 was used, and StudentNeumann-Keuls post hoc testing was performed for all pairwise comparisons. Statistics were performed using SigmaPlot (Systat Software, Chicago, IL) statistical software, version 12.0. All results are plotted as means ⫾ SE. RESULTS

Muscle weights and body weights. Table 1 summarizes the body weight and muscle weight-to body weight ratio of all groups receiving BM-MNC transplantation. All rats were 7 to 9 wk old at the beginning of the experiment. Most groups had a similar increase in body weight over the course of the experiment. The NSD ⫹ losartan BM-MNC treatment group

Table 1. Body and TA muscle weight-to-body weight ratio of all experimental groups receiving BM-MNC transplantation (BM-MNC donor rats not included) Body Weight, g BM-MNC Donor Diet

Recipient Diet

Route of Administration

Average Start Wt.

Average End Wt.

TA/BW, mg

NSD

NSD

Intramuscular

238 ⫾ 3.5

274 ⫾ 3.9*

NSD

HSD

Intramuscular

251 ⫾ 3.3

286 ⫾ 5.0*

HSD

HSD

Intramuscular

264 ⫾ 10

294 ⫾ 8.4*

NSD

HSD

Intravenous

243 ⫾ 1.5

277 ⫾ 3.8*

HSD

HSD

Intravenous

244 ⫾ 5.4

279 ⫾ 3.0*

HSD ⫹ saline

HSD

Intramuscular

245 ⫾ 2.8

289 ⫾ 4.3*

HSD ⫹ ANG II

HSD

Intramuscular

250 ⫾ 4.5

292 ⫾ 4.9*

NSD ⫹ losartan

HSD

Intramuscular

293 ⫾ 8.5

305 ⫾ 8.2

U: 1.85 ⫾ 0.04 S: 1.79 ⫾ 0.08 U: 1.75 ⫾ 0.05 S: 1.72 ⫾ 0.08 U: 1.67 ⫾ 0.09 S: 1.88 ⫾ 0.06 U: 1.71 ⫾ 0.11 S: 1.86 ⫾ 0.05 U: 1.77 ⫾ 0.04 S: 1.84 ⫾ 0.05 U: 1.75 ⫾ 0.03 S: 1.78 ⫾ 0.04 U: 1.75 ⫾ 0.03 S: 1.76 ⫾ 0.03 U: 1.80 ⫾ 0.03 S: 1.95 ⫾ 0.06

Values are means ⫾ SE. BM-MNC, bone marrow-derived mononuclear cell; NSD, normal-salt diet; HSD, high-salt diet; TA, tibialis anterior; Wt., weight; BW, body weight; U, unstimulated muscle; S, stimulated muscle. BW used to determine TA/BW ratio was the end average wt. of each animal. There were neither significant differences in the TA/BW between groups nor any effect of stimulation on muscle weight. *Significant difference vs. average start wt. (P ⱕ 0.05). AJP-Cell Physiol • doi:10.1152/ajpcell.00164.2013 • www.ajpcell.org

Fig. 2. A: changes in microvessel density in the tibialis anterior muscle from recipient NSD- and HSD-fed Sprague Dawley rats with an intramuscular injection in the stimulated limb (IMs) with either vehicle [Dulbecco’s phosphate-buffered saline without calcium or magnesium (DPBS); n ⫽ 6] or BM-MNCs from either NSD (n ⫽ 6) or HSD (n ⫽ 6) donor rats after 7 days of electrical stimulation. White bars, unstimulated; black bars, stimulated. Data shown as means ⫾ SE. *Significant difference (P ⱕ 0.05) vs. the unstimulated muscle. B: representative images of tibialis anterior muscle from unstimulated (U) and stimulated (S) limbs in each treatment group.

HIGH SALT INHIBITS BONE MARROW FUNCTION

A Microvessel Density (Vessel-Grid Intersections)

C126

B 180

Stimulated

*

*

140

HSD IMS Vehicle

120

HSD IMS NSD

100 80

HSD IMS NSD

Recipient Diet: NSD HSD HSD HSD Treatment: IMS Vehicle IMS Vehicle IMS NSD IMS HSD

did not have a significant increase in body; however, they were, on average, older than the other groups. There were no significant increases in TA muscle weight between the unstimulated and stimulated limbs. BM-MNC composition. BM-MNC composition was determined by light scatter analysis using flow cytometry (Fig. 1A). The total number of BM-MNCs per body weight isolated between the normal-salt and high-salt donors did not differ (379 ⫾ 31 thousand cells/g). There was a slight but significant decrease in the number of cells per body weight isolated from NSD ⫹ losartan treatment group (285 ⫾ 23 thousand cells/g). This observation is explained by a lower number of BM-MNCs isolated from the rats receiving this treatment (74 ⫾ 22 vs. 94 ⫾ 9 million cells in the other treatments) and not from a decrease in body weight. Light scatter analysis showed the population of BM-MNCs was composed of ⬃48.5% of lymphocyte sized cells and ⬃48.2% monocytes sized cells with relative few granulocyte sized cells (1.3%) in the NSD, HSD and NSD ⫹ losartan groups (Fig. 1B). However, the HSD ⫹ saline and HSD ⫹ ANG II BM-MNC treatment groups had a significant decrease in lymphocytes (33.4%), increase in monocytes (58.7%), and increased granulocytes (3.8%; Fig. 1B). This result is consistent with previous studies that examined BMMNC composition following surgical interventions. Attenuated therapeutic efficacy of high-salt BM-MNC transplantation. Vessel density following direct intramuscular injection of BM-MNCs into the stimulated TA was evaluated. NSD fed rats treated with vehicle (DPBS) had normal angio-

genesis following electrical stimulation, with a 31.8% increase in microvessel density between the unstimulated limb and the stimulated limb. HSD fed rats treated with vehicle did not undergo angiogenesis, with only an 8.6% [not significant (NS)] increase in microvessel density. Transplanted BM-MNCs from a NSD donor rat restored angiogenesis in HSD recipient rats, with a 24.2% increase in microvessel density. In contrast, BM-MNCs from HSD donors did not restore angiogenesis, with only a 6.0% (NS) increase in microvessel density (Fig. 2). A group of NSD recipients were also treated with NSD BM-MNCs. This treatment did not increase angiogenesis levels above what was seen in vehicle-treated NSD recipient rats (data not shown). Intravenous injection of normal-salt BM-MNCs restores angiogenesis. HSD SD rats were treated with a single bolus of 6 ⫻ 106 BM-MNCs injected into the tail vein (Fig. 3). BMMNCs from a normal-salt-fed donor were competent at increasing angiogenesis (10.3% increase, P ⫽ 0.007). BMMNCs from high-salt donors were incompetent at increasing angiogenesis in the angiogenic deficient recipient rats (HSD fed) with a 5.8% increase (NS). In vivo ANG II infusion of the donor rat restores angiogenic competency of HSD BM-MNCs. Donor SD rats on a HSD received 7 days of chronic jugular infusion of ANG II (3 ng·kg⫺1·min⫺1) or saline. These cells were transplanted by an intramuscular injection into HSD recipient rats. As shown in Fig. 4, A and B, ANG II-infusion confined only to the HSD

Microvessel Density

(Vessel-Grid Intersections)

A Fig. 3. A: comparison of microvessel density in the tibialis anterior of a HSD-fed SD recipient rat following tail vein intravenous injection (IV) of NSD BM-MNCs (n ⫽ 6) or HSD BM-MNCs (n ⫽ 6); 6 ⫻ 106 BM-MNCs were injected in a single bolus during the electrical stimulator surgery. Data shown as means ⫾ SE. *Significant difference vs. respective unstimulated limb (P ⱕ 0.05). B: representative images of tibialis anterior muscle from unstimulated (U) and stimulated (S) limbs in each treatment group.

S

NSD IMS Vehicle

Unstimulated

160

U

Unstimulated Stimulated 180

*

B HSD IV NSD

160 140

HSD IV HSD

120 100 80

Recipient Diet: Treatment:

HSD

HSD

IV NSD

IV HSD

AJP-Cell Physiol • doi:10.1152/ajpcell.00164.2013 • www.ajpcell.org

U

S

C127

HIGH SALT INHIBITS BONE MARROW FUNCTION

180

(Vessel-Grid Intersections)

Microvessel Density

A

B

Unstimulated

U

S

HSD IMS HSD+S

Stimulated 160

*

140

HSD IMS HSD+A

120 100 80

Recipient Diet: HSD Treatment: IMS HSD+S

HSD IMS HSD+A

D

C Microvessel Density

(Vessel-Grid Intersections)

180

Unstimulated

140

S

HSD IMS NSD

Stimulated

160

U

*

HSD IMS NSD+L

120

Fig. 4. Changes in microvessel density following modulation of the renin-angiotensin system in BM-MNC donor rats. Microvessel density is shown in the tibialis anterior after 7 days of electrical stimulation in SD rats on a HSD. A: direct intramuscular injection into the IMs of either 6 ⫻ 106 BM-MNCs from HSD saline infused (HSD ⫹ S, n ⫽ 7) donor rats or 6 ⫻ 106 BM-MNCs from high salt-fed donor rats infused with a low dose of ANG II (3 ng·kg⫺1·min⫺1, HSD ⫹ A, n ⫽ 7). B: representative images of tibialis anterior muscle from unstimulated (U) and stimulated (S) limbs in each treatment group. C: HSD recipient rats received a direct intramuscular injection of either 6 ⫻ 106 BM-MNCs from a donor rat on a NSD (NSD, n ⫽ 8) or 6 ⫻ 106 BM-MNCs from NSD rats with 50 mg/day losartan in the drinking water (NSD ⫹ L, n ⫽ 6). Note: 6 of the animals in the NSD BM-MNC group are the same animals as in Fig. 1. Data shown as means ⫾ SE. * Significant difference vs. respective unstimulated limb (P ⱕ 0.05). D: representative images of tibialis anterior muscle from unstimulated (U) and stimulated (S) limbs in each treatment group.

100 80

Recipient Diet: HSD IMS NSD Treatment:

HSD IMS NSD+L

donor rat restored angiogenic capabilities of BM-MNCs (13.4% increase, P ⱕ 0.05). Losartan treatment inhibits NSD BM-MNC angiogenic competency. Figure 4, C and D, shows that intramuscular injection of BM-MNCs derived from NSD-fed rats treated with losartan (50 mg/day, 5 days) failed to restore the angiogenic response in the TA muscle of HSD SD rats after 7 days of electrical stimulation. Losartan administration was confined to the donor animal only. Cleaved caspase-3 expression in BM-MNCs following hypoxic stimulus. To further understand the mechanisms involved in BM-MNC-mediated angiogenesis, we examined the protein expression of the apoptosis marker cleaved caspase-3 by immunoblot. Cells were treated with either normoxic (21% O2, 5% CO2) or hypoxic (2% O2, 5% CO2) incubation at 37°C for 6 h to mimic the effects of the ischemia seen in vivo. A PO2 of ⬃12 Torr was measured using an OM-4 oxygen meter (Microelectrodes) in the media of cell culture plates following 6 h of hypoxia treatment. Levels of cleaved caspase-3 were not different between BM-MNC treatment groups following a 6-h incubation at normoxic levels (Fig. 5). Following 6 h of hypoxia, HSD, HSD ⫹ saline, and NSD ⫹ losartan BM-MNCs all showed significant increases in caspase-3 cleavage between normoxic and hypoxic treatment. In vivo BM-MNC apoptosis following transplantation. We determined in vivo BM-MNC apoptosis following transplantation in skeletal muscle (Fig. 6); 15.4 ⫾ 2.2% of NSD BM-MNCs injected into the unstimulated limb were undergoing apoptosis. There was a slight but nonsignificant increase in

apoptosis of NSD BM-MNCs injected into the stimulated limb (28.7 ⫾ 2.9%). HSD BM-MNCs were apoptotic 20.6 ⫾ 6.4% in the unstimulated limb and had a significant increase in apoptosis in the stimulated limb 42.8 ⫾ 4.2%. Expression of HIF-1␣ and VEGF-A in BM-MNCs. HIF-1␣ and VEGF-A expressions were measured in isolated BMMNCs following a 6-h treatment at either normoxia or hypoxia. All treatments of BM-MNCs had similar increases in HIF-1␣ expression following hypoxia treatment of ⬃48 ⫾ 5%. However, only HSD ⫹ saline and NSD ⫹ losartan treatment groups reached significance due to large variations with in groups. Following 6 h of hypoxia, there was a significant increase in VEGF-A mRNA in NSD BM-MNCs (17.7% increase, P ⫽ 0.023) and NSD ⫹ losartan BM-MNCs (18.8% increase, P ⫽ 0.033) but not in HSD BM-MNCs (5.6% increase, NS), HSD ⫹ saline BM-MNCs (11.8% increase, NS), or HSD ⫹ ANG II BM-MNCs (0.3% increase, NS) compared with normoxic treatment. There were no significant differences between any of the treatment groups at baseline (6 h normoxia; Fig. 7). DISCUSSION

BM-MNC implantation can be used to revascularize tissue in angiogenic-deficient rats undergoing electrical stimulation. When donated from rats on a NSD, BM-MNCs were capable of restoring angiogenesis through both direct intramuscular or intravenous injection. The results of this study also suggest a critical and necessary role for circulating ANG II in mediating

AJP-Cell Physiol • doi:10.1152/ajpcell.00164.2013 • www.ajpcell.org

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A

C

H+S H+A + - +

Hypoxia: NA -

HSD - +

NSD - +

N+L - +

Cleaved Caspase3

19kDa 17kDa

Ponceau 19kDa 17kDa

B Cleaved Caspase3 (Arbitrary Units)

2 1.5

NSD+Losartan

* **

1

HSD HSD+Saline HSD+ANG II NSD

0.5 0

Normoxic Hypoxic Treatment of BM-MC

Fig. 5. A: representative immunoblot and corresponding Ponceau stain. B: quantitative densitometry comparing cleaved caspase-3 expression in BMMNCs of H ⫹ S (HSD ⫹ saline; n ⫽ 4), H ⫹ A (HSD ⫹ ANG II; n ⫽ 4), HSD (n ⫽ 3), NSD (n ⫽ 3), and N ⫹ L (NSD ⫹ losartan; n ⫽ 6) following either normoxic (21% O2, 5% CO2) or hypoxic (2% O2, 5% CO2) treatment. Data shown as means ⫾ SE. Samples were run on 3 individual blots and were normalized to Ponceau stain and to a positive control (C). *Significant difference vs. normoxic treatment (P ⱕ 0.05).

donor BM-MNC function. HSD suppressed BM-MNC angiogenic potential which was restored via a low-dose ANG II infusion. This pathway is likely acting through the angiotensin II receptor-1 (AT1R) pathway as demonstrated by the suppressed BM-MNC competency in losartan-treated rats. This dose of losartan has been shown to be effective to suppress skeletal muscle angiogenesis (2) and insufficient to inhibit

ANG II mediated increases in mean arterial blood pressure (15). Interestingly, the NSD BM-MNCs that were injected into normal angiogenic rats were not harmful nor were they capable of augmenting angiogenesis implicating a critical role of interaction of administered cells with the microenvironment of the angiogenic deficient stimulated skeletal muscle. HSD suppresses both renin (36) and ANG II (44). A lowdose infusion of ANG II, as used in this study, restores ANG II levels in the blood to the levels seen in NSD-fed rats (44) and concomitantly restores the angiogenic response in HSD rats. In the SD rat model used in this study, suppression of renin by HSD and the restoration of ANG II levels by low-dose infusion does not alter blood pressure (44). Thus all the results described are blood pressure independent. High-salt intake is associated with microvascular rarefaction, reduced tissue oxygen levels, and impaired muscle function (43) in both normotensive and hypertensive rats (22, 31). Under these conditions angiogenesis should cause a normalization of vessel density; however, this process also appears to be inhibited resulting in a lower steady-state vessel density with a HSD. Decreased capillary density induced by both rarefaction and an impaired angiogenic response leads to decreased hindlimb blood flow and impaired contraction of skeletal muscle (43). High-salt consumption has also been reported to directly impact arteriolar function, as arterioles harvested from rats fed a long-term HSD demonstrate a blunted dilatory response to a number of vasoactive stimuli (19). We have previously shown that changes in ANG II levels, as well as genetic manipulation of the renin gene, have profound effects on skeletal muscle angiogenesis induced by exercise or electrical stimulation (11, 13, 14). Here we show, for the first time, that HSD also impairs bone marrow function and that this impairment can be recovered by a low-dose administration of ANG II in the donor rat. Conversely, chronic high-dose ANG II infusion induces hypertension and has been associated with suppressed number of bone marrow derived stem cells and decreased cell function (25). Therefore, we suggest that normal ANG II levels are necessary for proper BM-MNC function. This study demonstrates a strong link between the suppression of the RAS by a HSD and dysfunction of BM-MNC.

A

Apoptotic BM-MNCs

Stimulated

(% Qdot Cells TUNEL positive)

Fig. 6. In vivo BM-MNC apoptosis. A: percentage of Quantum dot positive and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells following intramuscular injection into the unstimulated (Unstim) or stimulated (Stim) limb. Data shown as means ⫾ SE. #Significant difference vs. HSD Unstimulated. *Significant difference vs. NSD Unstimulated. (P ⱕ 0.05). B: representative images from 75-␮m thick sections of tibialis anterior muscle. TUNEL images have been corrected to remove background for publication only. Raw images were used for analysis. Magnification: ⫻40. Red: quantum dot (Qdot)-labeled BMMNCs. Green: apoptotic cells. Arrows: apoptotic and Qdot-positive cells.

B Unstimulated

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B Normoxic 10000 1000 100 10 1

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VEGF-A mRNA expression (mRNA compared to 18S)

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Fig. 7. A: mRNA for hypoxia-inducible factor-1␣ (HIF-1␣) relative to mRNA for ribosomal subunit 18S in BM-MNC. B: mRNA for VEGF-A relative to mRNA for ribosomal subunit 18S in BM-MNC. Cells were treated for 6 h at normoxia (21% O2, 5% CO2; n ⫽ 3) or hypoxia (2% O2, 5% CO2; n ⫽ 3). Data are means ⫾ SE.

1

Inhibition of the RAS by angiotensin-converting enzyme inhibitors or losartan impairs angiogenesis (1, 2). AT1R antagonists, such as losartan, are indicated to be used alone or in combination with other medications to treat high blood pressure and ensure renal protection and cardioprotection in humans. However, losartan also suppresses tumor-associated angiogenesis in a murine model of melanoma (40) and reduces microvessel reactivity to acetylcholine (56) potentially limiting blood flow to tissues. In this study we show that losartan treatment also impairs donor BM-MNC angiogenic capacity in normal-salt-fed SD rats exhibiting normal RAS. Cheng et al. (9) found that the AT1R antagonist Valsartan inhibited the angiogenic capacity of bone marrow stem cells. In that study they hypothesized that AT1R antagonists would alter the angiogenic activity of murine bone marrow stem cells in a hindlimb ischemia model. The discovery of AT1R expression in both murine (9) and rat (53) bone marrow cells provides growing evidence of activity of a local RAS in the bone marrow participating in hematopoiesis (20) and progenitor cell function. ANG II has also been shown to stimulate proliferation of the bone marrow hematopoietic progenitor cells (47), suggesting a broader role for the RAS in cardiovascular maintenance. Previously our laboratory has shown that HSD upregulates proapoptotic pathways in endothelial cells and in the skeletal muscle vasculature (12). In the present study, HSD caused increased apoptosis of BM-MNCs following a hypoxic stimulus. We measured the expression of cleaved caspase-3, a late apoptosis marker, following an acute bout of hypoxia in the BM-MNC of donor rats. BM-MNCs from HSD rats had higher levels of apoptosis than NSD BM-MNCs. Hypoxia is believed to be an important stimulus for angiogenesis and has been shown to increase expression of genes important for cell survival and proliferation in BM-MNCs (38). In recipient rats a HSD combined with electrical stimulation creates a low oxygen environment in the skeletal muscle (10). In such an environment, hypoxia can also induce apoptosis when the severity of hypoxia and the ability of the cell to adapt to the hypoxia triggers apoptosis either through the extrinsic pathway or through activation of the intrinsic mitochondria pathway (33). To confirm the physiological relevance of our in vitro observation we monitored BM-MNC apoptosis following injection into unstimulated and stimulated skeletal muscle in vivo. HSD BM-MNCs had a significant increase in apoptosis following electrical stimulation of the hindlimb muscles.

Since angiogenesis requires a complex interplay of multiple cell types and signals, decreased survival in vivo of HSD BM-MNCs may impact that interplay and be one mechanism by which HSD BM-MNCs are incapable of inducing angiogenesis. HIF-1␣ functions as a transcriptional regulator in response to hypoxia. Under hypoxic conditions, HIF-1␣ activates the transcription of ⬎40 genes, including vascular endothelial growth factor and other genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia. HIF-1␣ mRNA was increased similarly in all groups showing that all groups responded to hypoxia with increased HIF-1␣ expression. VEGF production has been shown to be induced in cells under hypoxic conditioning (24). In our studies, hypoxia increased VEGF-A mRNA expression in NSD and NSD ⫹ losartan BM-MNCs. However, there was no increase in VEGF-A expression in HSD, HSD ⫹ saline, or HSD ⫹ ANG II BM-MNCs. This suggests that VEGF-A expression in BMMNCs may be affected by dietary salt through a mechanism other than RAS modulation, because recovery of the RAS by ANG II infusion did not increase VEGF-A expression in the HSD rat. Similarly losartan treatment did not suppress VEGFA expression in the NSD rat. There are other proteins shown to be increased under hypoxic conditioning including other members of the VEGF family such as VEGF-B and VEGF-C (30), fibroblast growth factor family (28), and granulocyte-colony stimulating factor, a hematopoietic growth factor that increases mobilization of hematopoietic stem and progenitor cells from the bone marrow (49). These targets and others would be important to look into in future studies. The BM-MNC population is a complex mixed cell population including hematopoietic, mesynchmal, and endothelial progenitor cells. Each of these cell populations has been reported to promote or have effects on angiogenesis (4, 35, 39). Further studies characterizing each of the populations of cells within the BM-MNC fraction would be beneficial in elucidating the effect of dietary salt on these cells. It is also of interest to determine the effect of the transplanted BM-MNCs on the endogenous tissue following transplantation. Several questions including whether BM-MNCs directly participate in neovascularization and which specific cell type does this has yet to be determined. Determination of how BM-MNC transplantation mediates angiogenesis is the next hurtle in our understanding of this potential treatment option. The bone marrow is responsible for hematopoiesis and contains a variety of multipotent

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stem cells that can differentiate into an assortment of cell types including osteoblasts, chondrocytes, myocytes, adipocytes, and beta-pancreatic islets cells. A majority of bone marrow transplants are aimed at restoring normal bone marrow function in cancer patients, aplastic anemia, and sickle cell anemia patients. Since dietary salt affects the angiogenic capacity of the bone marrow as found in this study, it is reasonable to hypothesize HSD may affect other cell types and functions of the bone marrow. To our knowledge the effects of dietary salt have not been examined on hematopoiesis or on the other stem cell types in the bone marrow. In summary, the present study indicates that high dietary salt intake inhibits the angiogenic capacity of BM-MNCs via suppression of the RAS potentially limiting their therapeutic potential. Consideration of the states of the RAS and dietary salt intake of potential BM-MNC donors should be one consideration before BM-MNC harvest for therapeutics. ACKNOWLEDGMENTS We thank Daniela Didier and Timothy Stodola for technical assistance. We also thank Merck for the generous donation of losartan compound. GRANTS This study was funded by National Heart, Lung, and Blood Institute Grant HL-082798 (awarded to A. S. Greene) and also funded through Training Grant T32-HL-007852 (PI: H.V. Forster; awarded Department of Physiology at the Medical College of Wisconsin). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: J.R.K. conception and design of research; J.R.K. performed experiments; J.R.K. analyzed data; J.R.K. and A.S.G. interpreted results of experiments; J.R.K. prepared figures; J.R.K. drafted manuscript; J.R.K. and A.S.G. edited and revised manuscript; A.S.G. approved final version of manuscript. REFERENCES 1. Amaral SL, Linderman JR, Morse MM, Greene AS. Angiogenesis induced by electrical stimulation is mediated by angiotensin II and VEGF. Microcirculation 8: 57–67, 2001. 2. Amaral SL, Papanek PE, Greene AS. Angiotensin II and VEGF are involved in angiogenesis induced by short-term exercise training. Am J Physiol Heart Circ Physiol 281: H1163–H1169, 2001. 3. Antonios TF, Singer DR, Markandu ND, Mortimer PS, MacGregor GA. Rarefaction of skin capillaries in borderline essential hypertension suggests an early structural abnormality. Hypertension 34: 655–658, 1999. 4. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science 275: 964 –967, 1997. 5. Asahara T, Takahashi T, Masuda H, Kalka C, Chen D, Iwaguro H, Inai Y, Silver M, Isner JM. VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J 18: 3964 –3972, 1999. 6. Basford C, Forraz N, McGuckin C. Optimized multiparametric immunophenotyping of umbilical cord blood cells by flow cytometry. Nat Protoc 5: 1337–1346, 2010. 7. Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med 6: 389 –395, 2000. 8. Cheng C, Daskalakis C, Falkner B. Capillary rarefaction in treated and untreated hypertensive subjects. Ther Adv Cardiovasc Dis 2: 79 –88, 2008. 9. Cheng CI, Hsiao CC, Wu SC, Peng SY, Yip HK, Fu M, Wang FS. Valsartan impairs angiogenesis of mesenchymal stem cells through Akt pathway. Int J Cardiol, 2012. 10. Cornett JA, Herr MD, Gray KS, Smith MB, Yang QX, Sinoway LI. Ischemic exercise and the muscle metaboreflex. J Appl Physiol 89: 1432–1436, 2000.

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