The effects of young and aged, male and female megakaryocyte conditioned media on angiogenic properties of endothelial cells

Results

Proliferation of BMECs when cultured with MK CM

First, we examined cell growth as a proxy for the angiogenic potential of MK conditioned media (CM) on BMECs (Figure 1). While supplementation of young and aged male MK CM did not significantly impact the male BMEC cell number (Figure 1A), female BMEC proliferation was significantly improved with female MK CM, irrespective of age, compared to control female BMECs (Figure 1B). When treated with MK CM generated from mice of the opposite sex, male BMECs exhibited a significant increase in proliferation when cultured with young and old female MK CM (Figure 1D); female BMECs, however, only showed significantly greater proliferation when cultured with young male MK CM (Figure 1C). Together, these results suggest that female MKs secrete factors that increase both male and female BMEC proliferation. Notably, male MK CM generated from young mice significantly increased female BMEC proliferation and male MK CM generated from old mice showed a trending increase in female BMEC, whereas no significant or trending differences were observed when young or aged male MK CM was cultured with male BMECs. It should be noted that throughout this manuscript we report significant and trending differences due to the variability inherent in the use of primary cell cultures.

Figure 1. Proliferation of male and female BMECs increases with female MK CM treatment. (A) Male BMECs treated with male MK CM. (B) Female BMECs treated with female MK CM. (C) Female BMECs treated with male MK CM. (D) Male BMECs treated with female MK CM. The number of cells was quantified following a 48-hour incubation period. Data are expressed as a mean ± SD fold change relative to their respective controls (n = 3–11 biological replicates/group). For the negative control group (α-MEM lacking serum), n = 3 for male and 4 for female BMECs). Significance was determined using a one-way ANOVA with Tukey’s post-hoc test (A, B, D) or Kruskal-Wallis test (C) depending on the normalcy of the data distribution as determined by a Shapiro-Wilk test. Female BMECs treated with young female MK CM, old female MK CM, and young male MK CM proliferated significantly more than control female BMECs. Male BMECs treated with young or old female MK CM exhibited significantly greater proliferation. No significant differences in proliferation were found in male BMECs treated with male MK CM.

Effects of MK CM on vessel-like structure formation of BMECs

The effects of young and aged MK CM on the angiogenic potential of BMECs were evaluated by vessel-like structure formation (Figure 2). The criteria for what constitute a vessel-like structure are found in the Methods section. Vessel-like structure formation in the control and treatment groups was assessed and compared by the number of nodes, number of meshes, number of vessel-like structures, and total vessel length. As shown in Figure 2A–2H, treatment with young MK CM significantly improved all parameters in both males and females. Treatment with aged MK CM, irrespective of sex, significantly improved node and mesh numbers (Figure 2A–2D), and aged female MK CM significantly improved the number and length of vessel-like structures (Figure 2F, 2H, 2M–2O). On the other hand, aged male MK CM resulted in a trending, but not statistically significant, increase in the number of vessel-like structures (p = 0.22) and total vessel length (p = 0.14) (Figure 2E, 2G, 2I–2K). When comparing young and aged sex-matched MK CM, a significant difference between young and aged male MK CM on the number of vessel-like structures was detected (p = 0.044), where young male MK CM-treated groups had significantly more vessel-like structures than groups treated with aged male MK CM (Figure 2E). Images of positive control media (5% FBS) cultured with male and female BMECs are found in Figure 2L, 2P, respectively.

Figure 2. Various MK CM treatments increase length and complexity of BMEC vascular network. The number of nodes (A, B), the number of meshes (C, D), the number of vessel-like structures formed (E, F), and the total length of vessel-like structures (G, H) were quantified following an 8-hour incubation period. Representative images of BMEC vessel-like formation assays for control media (I, M), young MK CM (J, N), old MK CM (K, O), and positive control (L, P), with males in panels I-L, and females in panels (M–P). Data are expressed as a mean ± SD fold change relative to their respective controls (n = 8–13 biological replicates/group). Significance was determined using one-way ANOVA with Tukey’s post-hoc analysis (A, C, D, E) or Kruskal-Wallis test with Dunn’s post-hoc analysis (B, F, G, H) depending on the normalcy of the data distribution as determined by a Shapiro-Wilk test. Both young and aged female MK CM significantly increased all vessel-like formation properties. While young male MK CM also improved vessel-like properties, aged male MK CM did not increase the number of vessel-like structures nor the length of the vessel-like structures.

To examine how treatment of MK CM generated from mice of the opposite sex affects male and female BMEC tube formation, we also treated male BMECs with female MK CM from young or old mice and female BMECs with male MK CM from young or old mice (Figure 3). Interestingly, young and old male MK CM increased total vessel length of female BMECs and demonstrated a strong trend toward increasing total number of vessels (Figure 3A, 3B, 3E–3G). However, male BMECs did not respond to old female MK CM but did show a trend toward increased vessel number and length when treated with young female MK CM (Figure 3C, 3D, 3H–3J). Overall, the data in Figures 2 and 3 primarily support the idea that treatment of BMECs with MK CM, especially young MK CM, improves their capacity for vessel-like structure formation irrespective of the sex of the mice from which MKs were generated.

Figure 3. Young and old male MK CM significantly increase female BMEC vessel-like structure length. The number of vessel-like structures formed and total length of vessel-like structures were quantified following an 8-hour incubation period for female BMECs treated with male MK CM (A, B) and male BMECs treated with female MK CM (C, D). Representative images of BMEC vessel-like formation assays for Control media (E, H), young MK CM (F, I), and old MK CM (G, J), treatment groups are also shown for both female BMECs treated with male MK CM (E–G) and male BMECs treated with female MK CM (H–J). Data are expressed as a mean ± SD fold change relative to their respective controls (n = 4–8 biological replicates/group). Significance was determined using one-way ANOVA with Tukey’s post-hoc analysis (B–D) or Kruskal-Wallis test with Dunn’s post-hoc analysis (A) depending on the normalcy of the data distribution as determined by a Shapiro-Wilk test.

Effects of MK CM on BMEC unidimensional chemotaxis

The impacts of MK CM treatment on BMEC unidimensional chemotaxis were analyzed using transwell migration assays. As shown in Figure 4, the young male MK CM treatment elicited a trending increase in male BMEC migration, and the aged male MK CM significantly increased male BMEC migration (p = 0.047) compared to control male media (Figure 4A, 4C–4E). Similarly, female BMECs that were treated with aged female MK CM showed significantly more migration through the transwell membrane compared to control BMECs (p = 0.007, Figure 4B, 4G–4I). Images of positive control media (5% FBS) for male and female BMECs are found in Figure 4F, 4J, respectively. In general, aged MK CM promoted unidimensional migration of BMECs as compared to that observed with young MK CM.

Figure 4. Aged female and male MK CM increase unidimensional, transwell migration of BMECs. The number of migrated male BMECs (A) and female BMECs (B) was quantified following an overnight incubation period. Data are expressed as a mean ± SD fold change relative to their respective controls (n = 4–10 biological replicates/group). Significance was determined using a one-way ANOVA with Tukey’s post-hoc test. Aged MK CM significantly increased the migration of BMECs compared to control regardless of sex. Representative images of male BMECs using crystal violet staining (C- Control, D- Young Male MK CM, E- Old Male MK CM, F- 5% FBS control). Representative images of female BMECs using crystal violet staining (G- Control, H- Young Female MK CM, I- Old Female MK CM, J- 5% FBS control).

Effects of MK CM on BMEC two-dimensional motility

The effects of young and aged MK CM treatment on BMEC two-dimensional motility were examined through a wound migration assay, which compared the treatment and control groups in terms of the relative wound density of cells within the produced wound and the wound width over 48 hours (Figure 5). While relative wound density increased over time for all 3 groups, male MK CM treatment in male BMECs and control treatment in female BMECs led to greater relative wound density when compared by 2-way ANOVA; no significant differences were observed between groups specifically at 12, 24, or 48 hours (Figure 5A, 5B). When wound width was examined, control media resulted in the greatest decrease in both male and female BMECs compared to the MK CM groups, compared by 2-way ANOVA (Figure 5C, 5D). Similarly, no significant differences were observed between the groups specifically at 12, 24, or 48 hours for wound width in males. However, there was a significant difference between aged female and control wound width (p = 0.029) at 48 hours.

Figure 5. Altered two-dimensional motility of BMECs with MK CM treatments. The changes in relative wound density (A-males, B-females) and wound width (C-males, D-females) were quantified through data collected every 2 hours over a 48-hour period. Significance was determined using a two-way ANOVA with Dunnett’s post-hoc test (significance bars are noted to the right on all panels). Cross-sectional analyses were made at 12-, 24-, and 48-hour timepoints for all panels as well. No significant differences in relative wound density (A, B) were observed in any cross-sectional analyses. A one-way ANOVA found that female BMEC wound closure (panel D) was significantly inhibited when treated with female aged MK CM at the 48-hour timepoint (p = 0.029), but all other cross-sectional analyses were not significant in panels C and D. Solid, dashed, and dotted lines represent the means (n = 3–16 biological replicates/group).

MK CM alters gene expression of angiogenic and inflammatory factors in BMECs

Further understanding of how MK CM affects BMECs was pursued. While the previous experiments analyzed BMEC functionality on a cellular level, we sought to examine the molecular changes as well. Eleven genes of receptors present in BMECs were examined for their relative mRNA expression (Figure 6 and Supplementary Figure 1). The selection process was based on the known presence of specific factors in MK CM. These factors may bind to the gene’s encoded receptors and have a downstream effect of changing the degree of transcription, thereby influencing the proangiogenic and antiangiogenic properties of the BMECs [44, 45].

Figure 6. Certain MK CM treatments alter various genes of BMECs to a proangiogenic and proinflammatory phenotype. The changes in mRNA expression were quantified following an overnight incubation period. Data are expressed as a mean ± SD fold change relative to their respective controls with post-hoc p-values listed in each panel (n = 3–4 biological replicates/group). GAPDH was used as an internal control. Significance was determined using one-way ANOVA with Tukey’s post-hoc analysis or Kruskal-Wallis test with Dunn’s post-hoc analysis depending on the normalcy of the data distribution as determined by a Shapiro-Wilk test. MK CM treatments showed trending or significant increases in BMEC expression of CXCR2 (A, B) and PDGFRβ (E, F), while showing trending or significant decreases of CD36 (G, H), CD74 (I, J), and TGFβR2 (K, L) expression. Although aged male and young female MK CM significantly increased CXCR4 expression, young male and aged female MK CM did not (C, D). No significant differences in gene expression levels were seen in BMPR2 (M, N) or CD47 (O, P).

Of the eleven genes examined, five were found to have at least one type of MK CM exhibit significantly altered expression relative to controls. Both young and old male MK CM increased BMEC expression of CXCR2 (Figure 6A) though not significantly. Young female MK CM significantly increased CXCR2 expression, relative to both control and aged female MK CM (Figure 6B). Aged male MK CM significantly increased expression of CXCR4 (Figure 6C) not only relative to control, but also compared to young male MK CM. The opposite was true for CXCR4 with female MK CM, as young female MK CM significantly increased its expression relative to both control and aged female MK CM treatment (Figure 6D). Aged female MK CM increased expression of PDGFRβ to a nonsignificant degree, which was found to have mirroring trends in young female and young male MK CM treatments for this gene (Figure 6E, 6F). Young and aged female MK CM significantly decreased expression of CD36, while male MK CM showed trending decreases (Figure 6G, 6H). Aged male MK CM reduced expression of CD74 to a significant extent, while young male, young female, and old female MK CM treatments showed no significant change in expression of CD74 (Figure 6I, 6J). Female MK CM, both young and aged, decreased expression of TGFβR2 significantly while male MK CM treatments decreased its expression to a nonsignificant degree (Figure 6K, 6L). BMPR2 and CD47 expression remained unchanged (Figure 6M–6P), as did all other genes in Supplementary Figure 1.

Materials and Methods

Animals

Young (3–4-month-old) and old (22–24-month-old), male and female C57BL/6J mice were purchased from Jackson Laboratory or were generously provided by the National Institute of Aging (NIA), respectively. The Indiana University School of Medicine Institutional Animal Care and Use Committee approved all described studies.

EC isolation from BM

BMECs were isolated from the BM of femurs, tibiae, and humeri of all mice. Briefly, after euthanasia, skin, muscle, and soft tissue were stripped from each of the bones, after which the distal and proximal epiphyses of the bones were removed. All bones were individually placed into sterile, punctured 0.5 mL tubes that were inserted into 1.5 mL tubes. Each 1.5 mL tube contained 750 μL α-MEM (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS, Biowest, Riverside, MO, USA). BM was isolated from the bones by centrifugation at 14,000 g for 2 minutes at 4°C. The extracted BM pellets were then resuspended in 1 mL of Complete EC Growth Media (ScienCell, Carlsbad, CA, USA), which contained EC growth supplements, 7.5% FBS, and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA). The resuspended pellets were plated in a 12-well plate coated with 4 μg/mL fibronectin (Thermo Fisher Scientific, Waltham, MA, USA) containing an additional 1 mL of Complete EC Growth Media. Media changes were performed every two days and BMECs were cultured for 5–7 days prior to use in experimental assays.

MK CM preparation

Murine BM from C57BL/6J mice was extracted and plated in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA), supplemented with 5% FBS (Hyclone, Logan, UT, USA), 1% penicillin/streptomycin/glutamine (Gibco, Grand Island, NY, USA), and conditioned medium from a TPO producing cell line [38] until numerous large MKs were observed. MKs were then isolated from the other cell population using a one-step Bovine Serum Albumin (BSA, Millipore-Sigma, Burlington, MA, USA) gradient [38]. The isolated MKs were removed from the bottom of the gradient, washed with Phosphate Buffered Saline (PBS, Gibco, Grand Island, NY, USA), and re-suspended in phenol-free α-MEM at 1 million cells/ml for 3 days. The CM was collected, centrifuged, and stored at −80°C until use.

Proliferation assay

BMECs were seeded in a 96-well plate (Corning, Corning, NY, USA) at 1 × 10³ cells/well with 25% MK CM isolated from young or old mice of both sexes (all containing 1.25% FBS), or 25% α-MEM (Control Media) containing 1.25% FBS, or α-MEM with 0% FBS (Negative control) and were incubated for 1 to 2 days to assess proliferation. The BMECs were fixed with 5% neutral buffered formalin (NBF) at room temperature for 15 minutes. The fixed BMECs were stained with 0.05% crystal violet for 20 minutes and were subsequently washed with tap water prior to an overnight drying period. The EVOS FL Cell Imaging System was utilized to image BMEC cultures, and ImageJ.1.52a software was used for counting analysis.

Vessel-like structure formation assay

96-well plates (Corning Inc., Corning, NY, USA) were chilled in a −20°C freezer overnight. They were subsequently coated with 50 μL/well of Matrigel basement membrane matrix (Corning, Corning, NY, USA) and incubated 45 minutes at 37°C prior to plating and culturing BMECs. BMECs were seeded at 1 × 104 cells/well with their respective treatments and incubated for 8 hours prior to cell imaging (EVOS FL Cell Imaging System). α-MEM (Control group), 25% MK CM isolated from young or old mice of both sexes (all containing 1.25% FBS), and α-MEM with 5% FBS (positive control) were the designated groups. Vessel-like structure formation was quantified with ImageJ.1.52a software. Images are taken at 10× magnification. The number of nodes, number of meshes, number of complete vessels, and total vessel-like structure length were the parameters analyzed and an example is shown in Figure 7 [30]. Briefly, meshes were defined as areas enclosed by three or more nodes, and nodes were defined as a point where two or more vessels intersect. Simple Neurite Tracer, a plugin of ImageJ.1.52b software, was employed for the manual measurement of the total vessel length and number of vessel-like structures. A blinded analyzer manually measured the number of vessel-like structures as well as vessel length. The automated Angiogenesis Analyzer, a plugin of ImageJ.1.52a software, was used to analyze the number of meshes and nodes.

Figure 7. Representative image of BMEC vessel-like formation assay. Images were taken at 10x magnification. Meshes (red outline) and nodes (red star) were automatically analyzed. Meshes were defined as areas enclosed by three or more nodes and nodes were defined as a point where two or more vessels intersect. A blinded analyzer manually measured the number of vessel-like structures (indicated by blue arrows) as well as vessel length.

Transwell assay or unidimensional chemotaxis assay

500 μL of 25% MK CM or 25% α-MEM was added to the bottom of each well in 24-well plates; α-MEM with 5% FBS was used as a positive control. A transwell membrane (8 μm pore size, Corning, Corning, NY, USA) was placed over the media. Cells resuspended in Basal EC Growth Media (ScienCell, Carlsbad, CA, USA containing only 7.5% FBS and 1% penicillin/streptomycin solution. Basal EC Growth Media lacked EC growth supplements.) were seeded on the top of the membrane at a density of 4 × 10⁵ cells/well. The plates were placed in an incubator at 37°C for a period of 24 hours. The membranes were fixed with 5% NBF at room temperature for 15 minutes. The membranes were stained with 0.5% crystal violet for 20 minutes and dried using sterile cotton tip applicators (Puritan Medical Products, Gulliford, ME, USA). The EVOS FL Cell Imaging System was used to image the migration of the BMECs across the membrane at a magnification of 10×. Analysis of images was completed with the ImageJ.1.52a software.

Wound migration assay

96-well plates (Corning, Corning, NY, USA) were seeded with BMECs placed in Complete EC Growth Media at a density of 1 × 10⁵ cells/well and incubated at 37°C for a period of 24 hours to achieve 100% confluence. Complete EC Growth Media was aspirated and replaced with 25% MK CM or 25% α-MEM. The IncuCyte^® WoundMaker (Essen BioScience, Ann Arbor, MI, USA) generated a wound in the center of every well. The IncuCyte ZOOM^® Live-Cell Analysis System (Essen BioScience, Ann Arbor, MI, USA) took images of each well every 2 hours at 10X magnification for 48 hours. Relative wound density and wound widths were compared across the entire timecourse and specifically at 12, 24, and 48 hours. The IncuCyte^™ Scratch Wound Cell Migration Software (Essen BioScience, Ann Arbor, MI, USA) analyzed the images taken.

RNA isolation and gene expression

BMECs were seeded in a 6-well plate at a concentration of 1 × 10⁶ cells/well with 25% MK CM or 25% α-MEM. The plates were incubated at 37°C for 24 hours to achieve no less than 80% confluency. The RNeasy Mini kit (QIAGEN, Hilden, Germany) was used for total RNA isolation. The Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) was used to prepare cDNA, which was then analyzed through quantitative PCR using the Power SYBR^™ Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). GAPDH was used as an internal control. The fold change in gene expression was obtained through the 2^−ΔΔCT method. Several genes were analyzed. Table 2 lists the genes and primers used.

Table 2. Primer pairs for gene expression.

Gene	Forward sequence	Reverse sequence
CXCR2	CTCTATTCTGCCAGATGCTGTCC	ACAAGGCTCAGCAGAGTCACCA
CXCR4	GACTGGCATAGTCGGCAATGGA	CAAAGAGGAGGTCAGCCACTGA
PDGFRβ	CATCCGCTCCTTTGATGATCTT	GTGCTCGGGTCATGTTCAAGT
CD36	GGACATTGAGATTCTTTTCCTCTG	GCAAAGGCATTGGCTGGAAGAAC
CD74	CGCGACCTCATCTCTAACCAT	ACAGGTTTGGCAGATTTCGG
TGFβR2	CCTACTCTGTCTGTGGATGACC	GACATCCGTCTGCTTGAACGAC
BMPR2	AGAGACCCAAGTTCCCAGAAGC	TCTCCTCAGCACACTGTGCAGT
CD47	TGCGGTTCAGCTCAACTACTG	GCTTTGCGCCTCCACATTAC
ITGβ1	CTCCAGAAGGTGGCTTTGATGC	GTGAAACCCAGCATCCGTGGAA
FLT1	CCACCTCTCTATCCGCTGG	ACCAATGTGCTAACCGTCTTATT
KDR/VEGFR2	TTTGGCAAATACAACCCTTCAGA	GCTCCAGTATCATTTCCAACCA
GAPDH	CGTGGGGCTGCCCAGAACAT	TCTCCAGGCGGCACGTCAGA

Statistics

Statistical analyses and data normality calculations were performed with GraphPad Prism (ver. 8 and 10, GraphPad Software, La Jolla, CA, USA). Results are displayed in figures as mean ± SD or line graphs. Data in this manuscript comparing 3 or more groups were analyzed by one-way analysis of variance (ANOVA) with Tukey-Kramer post hoc test for parametric data. A Kruskal-Wallis test with a Dunn’s multiple comparison post-hoc test was used for nonparametric data. For the wound healing assay, a two-way ANOVA with a Dunnett’s Multiple comparison test was used to examine the main treatment effects. Specific post-hoc p-values are displayed on the graphs, and a p-value of less than 0.05 was considered significant.

Abbreviations

BM: bone marrow; BMEC: bone marrow endothelial cell; BMP: bone morphogenetic protein; BSA: bovine serum albumin; CM: conditioned media; DMEM: Dulbecco’s Modified Eagle Medium; EC: endothelial cell; ENA: epithelial neutrophil-activating peptide; FBS: fetal bovine serum; FGF: fibroblast growth factor; GRO: growth-related oncogenes; HIF: hypoxia-inducible factor; IL: interleukin; MIF: migration inhibitory factor; MK: megakaryocyte; NAP: neutrophil-activating peptide; NBF: neutral buffered formalin; NIA: National Institute of Aging; OPG: osteoprotegerin; PDGF: platelet-derived growth factor; SDF: stromal-cell derived factor; SIRT1: sirtuin 1; TGF: transforming growth factor; TPO: thrombopoietin; VEGF: vascular endothelial growth factor.

Author Contributions

M.K. Nazzal, H.L. Battina, N.P. Tewari, U.C. Dadwal, and M.A. Kacena designed the research; M.K. Nazzal, H.L. Battina, N.P. Tewari, S.L. Mostardo, R.U. Nagaraj, D. Zhou, O.D. Awosanya, S.K. Majety, S. Samson, R.J. Blosser, U.C. Dadwal, and P.L. Mulcrone performed the research; M.K. Nazzal, H.L. Battina, N.P. Tewari, and M.A. Kacena wrote the paper; M.K. Nazzal, H.L. Battina, N.P. Tewari, S.L. Mostardo, R.U. Nagaraj, D. Zhou, O.D. Awosanya, S.K. Majety, S. Samson, R.J. Blosser, P.L. Mulcrone, U.C. Dadwal, and M.A. Kacena analyzed the data, critically revised the paper, approved the final version of the paper, and take responsibility for the contents of the paper. M.A. Kacena and P.L. Mulcrone take responsibility for the integrity of the data analysis.

Acknowledgments

We would like to thank the Indiana University Angiogenesis Core for aiding with wound migration studies. We would also like to thank the Indiana University In Vitro Therapeutics Cores for providing mice used for these studies.

Conflicts of Interest

The authors declare no conflicts of interest related to this study.

Ethical Statement

The Indiana University School of Medicine Institutional Animal Care and Use Committee approved all described studies, protocol number is 23133.

Funding

Core facilities are supported in part by the NIH U24DK126127. This project was financed, in part, by the Cooperative Center of Excellence in Hematology (CCEH) Award, funded by the National Institute of Diabetes and Digestive and Kidney Diseases of the NIH under U24DK126127 (MAK, UCD) and U54DK106846 (NPT). The project was also supported, in part, by NIH T32 DK007519 (UCD), NIH T32 AR065971 (UCD), NIH R01 AG060621 (MAK), NIH T32 AR065971 (MKN), NIH F31 AG077931 (ODA), and NIH T32 HL007910 (ODA). In addition, the results of this work were supported with resources and the use of facilities at the Richard L. Roudebush VA Medical Center, Indianapolis, IN: VA Merit #BX003751 and #RX003552 (MAK) and Research Career Scientist #IRX004809A. The views expressed in this article are solely those of the authors and do not necessarily represent the official position or policy of any of the agencies mentioned.

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