J Pathol Transl Med.  2015 Nov;49(6):472-480. 10.4132/jptm.2015.09.11.

Therapeutic Effects of Umbilical Cord Blood Derived Mesenchymal Stem Cell-Conditioned Medium on Pulmonary Arterial Hypertension in Rats

• Lee JC
• Cha CI
• Kim DS
• Choe SY

• KMID: 2381406
• DOI: http://doi.org/10.4132/jptm.2015.09.11

Abstract

BACKGROUND
Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) may have multiple therapeutic applications for cell based therapy including the treatment of pulmonary artery hypertension (PAH). As low survival rates and potential tumorigenicity of implanted cells could undermine the mesenchymal stem cell (MSC) cell-based therapy, we chose to investigate the use of conditioned medium (CM) from a culture of MSC cells as a feasible alternative.
METHODS
CM was prepared by culturing hUCB-MSCs in three-dimensional spheroids. In a rat model of PAH induced by monocrotaline, we infused CM or the control unconditioned culture media via the tail-vein of 6-week-old Sprague-Dawley rats.
RESULTS
Compared with the control unconditioned media, CM infusion reduced the ventricular pressure, the right ventricle/(left ventricle+interventricular septum) ratio, and maintained respiratory function in the treated animals. Also, the number of interleukin 1alpha (IL-1alpha), chemokine (C-C motif) ligand 5 (CCL5), and tissue inhibitor of metalloproteinase 1 (TIMP-1)-positive cells increased in lung samples and the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique (TUNEL)-positive cells decreased significantly in the CM treated animals.
CONCLUSIONS
From our in vivo data in the rat model, the observed decreases in the TUNEL staining suggest a potential therapeutic benefit of the CM in ameliorating PAH-mediated lung tissue damage. Increased IL-1alpha, CCL5, and TIMP-1 levels may play important roles in this regard.

Keyword

Apoptosis; Culture media, conditioned; Gene expression; Mesenchymal stromal cells; Pulmonary artery hypertension

MeSH Terms

Animals
Apoptosis
Culture Media
Culture Media, Conditioned
Deoxyuridine
Fetal Blood*
Gene Expression
Humans
Hypertension*
In Situ Nick-End Labeling
Interleukin-1alpha
Lung
Mesenchymal Stromal Cells
Models, Animal
Monocrotaline
Pulmonary Artery
Rats*
Rats, Sprague-Dawley
Survival Rate
Tissue Inhibitor of Metalloproteinase-1
Umbilical Cord*
Ventricular Pressure
Culture Media
Culture Media, Conditioned
Deoxyuridine
Interleukin-1alpha
Monocrotaline
Tissue Inhibitor of Metalloproteinase-1

Figure

• 

  Fig. 1. Inflammatory cytokine expressions in the three groups. (A) To screen whether hUCB-MSCs-CM affect local production of inflammatory cytokines by lung cells in the three groups, a cytokine array is performed on lung homogenates. (B) TIMP-1, IL-1α, and CCL5 are higher in the CM group compared to the C and M groups, whereas CCL7 and VEGF are lower in the CM group compared to the M group. CINC-1, CINC-2a/b, CX3CL1 LIX, and LECAM-1 are higher in the C group compared to the M and CM groups. C group, control group (n = 7); M group, monocrotaline group (n=7); CM group, hUCB-MSCs-CM group (n=7). hUCB-MSCs-CM, conditioned medium from human umbilical-cord blood derived mesenchymal cells; CINC-1, cytokine-induced neutrophil chemoattractant-1; CINC-2a/b, cytokine-induced neutrophil chemoattractant-2a/b; CX3CL1, chemokine (C-X-C motif) ligand 1; LIX, lipopolysaccharide-induced CXC chemokine; LECAM-1, leukocyte endothelial cell adhesion molecule 1; CCL7, chemokine (C-C motif) ligand 7; TIMP-1, tissue inhibitor of metalloproteinase 1; VEGF, vascular endothelial growth factor; IL-1α, interleukin 1α. ap<.05 compared with the C group; b,cp<.05 compared with the M group.

• 

  Fig. 2. Localization of IL-1α, CCL5, and TIMP-1-immunoreactive cells in the lung tissues at 28 days. (A–R) Immunohistochemical expression reveals that the positive cells of IL-1α, CCL5, and TIMP-1 are significantly higher in the CM group than that in the C and M groups, and they are higher in the M group than that in the C group. (S) The increased levels of IL-1α, CCL5, and TIMP-1 immunoreactivity observed in the CM group are statistically significant. The levels of IL-1α, CCL5, and TIMP-1 immunoreactivity are significantly decreased in the CM group compared with the C and M groups. Panels A–C, G–I, and M–O are high power views of panels D–F, J–L, and P–R, respectively. C, control; M, monocrotaline; CM, hUCB-MSCs-CM; hUCB-MSCs-CM, conditioned medium from human umbilical-cord blood derived mesenchymal cells; TIMP-1, tissue inhibitor of metalloproteinase 1; IL-1α, interleukin 1α; CCL5, chemokine (C-C motif) ligand 5. ap<.05 compared with the C group; bp<.05 compared with the M group.

• 

  Fig. 3. Changes of IL-1α, CCL5, and TIMP-1 protein expression levels after hUCB-MSCs-CM injection in PAH rats. (A) These are pictures of protein expression levels of IL-1α, CCL5, and TIMP-1 in the lung tissues. (B) The protein expressions levels of IL-1α, CCL5, and TIMP-1 at 28 days are significantly increased in the CM group compared to the C and M groups. The protein expressions of CCL are increased in the M group compared to the C group. C, control; M, monocrotaline; CM, hUCB-MSCs-CM; hUCB-MSCs-CM, conditioned medium from human umbilical-cord blood derived mesenchymal cells; TIMP-1, tissue inhibitor of metalloproteinase 1; IL-1α, interleukin 1α; CCL5, chemokine (C-C motif) ligand 5. ap<.05 compared with the C group; bp<.05 compared with the M group.

• 

  Fig. 4. Changes of caspase-3 and Bcl-2 protein expression levels after hUCB-MSCs-CM injection in PAH rats. (A) These are pictures of protein expression levels of caspase-3 and Bcl-2 in the lung tissues. (B) The protein expressions levels of caspase-3 and Bcl-2 at 28 days are significantly increased in the M group compared to the C groups. However, the protein expressions levels of caspase-3 and Bcl-2 are decreased in the CM group compared to the M group. C, control; M, monocrotaline; CM, hUCB-MSCs-CM; hUCB-MSCs-CM, conditioned medium from human umbilical-cord blood derived mesenchymal cells. ap<.05 compared with the C group; bp<.05 compared with the M group.

• 

  Fig. 5. TUNEL assay of lung tissues at 28 days after hUCB-MSCs-CM transfusion. (A–F) Immunohistochemical expression reveals that the positive cells of apoptosis are significantly higher in the M group than that in the C group; however, they are lower in the CM group than that in the M group. (G) The increased levels of TUNEL immunoreactivity observed in the M group are statistically significant. The levels of TUNEL immunoreactivity are significantly decreased in the CM group compared with the M group. This result indicates that hUCB-MSCs-CM could attenuate the vascular remodeling. Panels A–C are high power views of panels D–F, respectively. C, control; M, monocrotaline; CM, hUCBMSCs-CM; hUCB-MSCs-CM, conditioned medium from human umbilical-cord blood derived mesenchymal cells. ap<.05 compared with the C group; bp<.05 compared with the M group.

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