Janice Arakawa-Hoyt

CD57 defines a functionally distinct population of mature NK cells in the human CD56dimCD16+ NK-cell subset

Natural killer (NK) cells are innate immune lymphocytes that express a heterogeneous repertoire of germline-encoded receptors and undergo a distinct pattern of maturation. CD57 is a marker of terminal differentiation on human CD8(+) T cells. Very few newborn or fetal NK cells express CD57; however, the frequency of CD57-bearing NK cells increases with age. We assessed the transcriptional, phenotypic, and functional differences between CD57(+) and CD57(-) NK cells within the CD56(dim) mature NK subset. CD57(+) NK cells express a repertoire of NK-cell receptors, suggestive of a more mature phenotype, and proliferate less when stimulated with target cells and/or cytokines. By contrast, a higher frequency of CD57(+) NK cells produced interferon-γ and demonstrated more potent lytic activity when these cells were stimulated through the activating receptor CD16; however, they are less responsive to stimulation by interleukin-12 and interleukin-18. Finally, CD57 expression is induced on CD57(-)CD56(dim) NK cells after activation by interleukin-2. A combination of a mature phenotype, a higher cytotoxic capacity, a higher sensitivity to stimulation via CD16, with a decreased responsiveness to cytokines, and a decreased capacity to proliferate suggest that CD57(+) NK cells are highly mature and might be terminally differentiated.

Adeno-associated viral vector-mediated hypoxia response element-regulated gene expression in mouse ischemic heart model

Intramyocardial injection of genes encoding angiogenic factors could provide a useful approach for the treatment of ischemic heart disease. However, uncontrolled expression of angiogenic factors in vivo may cause some unwanted side effects, such as hemangioma formation, retinopathy, and arthritis. It may also induce occult tumor growth and artherosclerotic plaque progression. Because hypoxia-inducible factor 1 is up-regulated in a variety of hypoxic conditions and it regulates gene expression by binding to a cis-acting hypoxia-responsive element (HRE), we propose to use HRE, found in the 3′ end of the erythropoietin gene to control gene expression in ischemic myocardium. A concatemer of nine copies of the consensus sequence of HRE isolated from the erythropoietin enhancer was used to mediate hypoxia induction. We constructed two adeno-associated viral vectors in which LacZ and vascular endothelial growth factor (VEGF) expressions were controlled by this HRE concatemer and a minimal simian virus 40 promoter. Both LacZ and VEGF expression were induced by hypoxia and/or anoxia in several cell lines transduced with these vectors. The functions of these vectors in ischemic myocardium were tested by injecting them into normal and ischemic mouse myocardium created by occlusion of the left anterior descending coronary artery. The expression of LacZ gene was induced eight times and of VEGF 20 times in ischemic myocardium compared with normal myocardium after the viral vector transduction. Hence, HRE is a good candidate for the control of angiogenic factor gene expression in ischemic myocardium.

VEGF improves survival of mesenchymal stem cells in infarcted hearts

Bone marrow-derived mesenchymal stem cells (MSC) are a promising source for cell-based treatment of myocardial infarction (MI), but existing strategies are restricted by low cell survival and engraftment. We examined whether vascular endothelial growth factor (VEGF) improve MSC viability in infarcted hearts. We found long-term culture increased MSC-cellular stress: expressing more cell cycle inhibitors, p16(INK), p21 and p19(ARF). VEGF treatment reduced cellular stress, increased pro-survival factors, phosphorylated-Akt and Bcl-xL expression and cell proliferation. Co-injection of MSCs with VEGF to MI hearts increased cell engraftment and resulted in better improvement of cardiac function than that injected with MSCs or VEGF alone. In conclusion, VEGF protects MSCs from culture-induce cellular stress and improves their viability in ischemic myocardium, which results in improvements of their therapeutic effect for the treatment of MI.

AAV serotype-1 mediates early onset of gene expression in mouse hearts and results in better therapeutic effect

Adeno-associated viral vectors (AAV) are attractive tool for gene therapy for coronary artery disease. However, gene expression in myocardium mediated by AAV serotype 2 (AAV2) does not peak until 4–6 weeks after gene transfer. This delayed gene expression may reduce its therapeutic potential for acute cardiac infarction. To determine whether earlier gene expression and better therapeutic effect could be achieved using a different serotype, CMV promoter driving the EPO gene (AAV-EPO) was packaged into AAV serotypes 1–5 capsids and injected into mouse myocardium. EPO expression was studied by measuring the hematocrits and EPO mRNA. After we found that AAV1 mediates the highest gene expression after 4 days of gene transduction, AAV-LacZ (CMV promoter driving LacZ gene expression) and MLCVEGF (hypoxia-inducible and cardiac-specific VEGF expression) were packaged into AAV1 and 2 capsids. LacZ expression was detected in AAV1-LacZ but not in AAV2-LacZ-injected hearts 1 day after vector injection. Compared to AAV2-MLCVEGF that mediated no significant VEGF expression, AAV1-MLCVEGF mediated 13.7-fold induction of VEGF expression in ischemic hearts 4 days after gene transduction and resulted in more neovasculatures, better cardiac function and less myocardial fibrosis. Thus, AAV1 mediates earlier and higher transgene expression in myocardium and better therapeutic effects.

Combining angiogenic gene and stem cell therapies for myocardial infarction

Transplantation of stem cells from various sources into infarcted hearts has the potential to promote myocardial regeneration. However, the regenerative capacity is limited partly as a result of the low survival rate of the transplanted cells in the ischemic myocardium. In the present study, we tested the hypothesis that combining cell and angiogenic gene therapies would provide additive therapeutic effects via co‐injection of bone marrow‐derived mesenchymal stem cells (MSCs) with an adeno‐associated viral vector (AAV), MLCVEGF, which expresses vascular endothelial growth factor (VEGF) in a cardiac‐specific and hypoxia‐inducible manner.

AAV serotype 1 mediates more efficient gene transfer to pig myocardium than AAV serotype 2 and plasmid.

Adeno‐associated virus (AAV) has many properties of an ideal vector for delivery of therapeutic genes into the myocardium. Previous studies in a mouse model of myocardial infarction showed that AAV serotype 1 (AAV1) is superior to AAV serotypes 2–5 to transfer genes into the myocardium by direct injection. Since vectors may behave differently in humans and because the human and the pig hearts resemble each other closely, we tested whether AAV1 is also superior to AAV2 in transferring genes into the pig myocardium. We also compared gene transduction efficiency between AAV vectors and plasmid. We injected CMVLacZ and CMVVEGF (vectors with the cytomegalovirus (CMV) promoter driving LacZ and VEGF gene expression) unpackaged or packaged in AAV serotypes 1 or 2 capsids into pig myocardium. Hearts were collected 3, 14 and 28 days after the injection. Gene expression was analyzed by real‐time reverse‐transcription polymerase chain reaction (RT‐PCR) and histological staining. Capillaries and smooth muscle α‐actin (SMA)‐positive vessels were quantified. Potential lymphocyte infiltration at the injection sites was analyzed by immunostaining using specific antibodies. As in the mouse, AAV1 mediated better gene transduction than AAV2. Plasmid mediated minimal gene expression only. More capillaries and SMA‐positive vessels were detected at AAV1CMVVEGF‐ and AAV2CMVVEGF‐injected than AAV1CMVLacZ‐injected sites. We did not detect inflammatory cell infiltration at the injection sites. In conclusion, by direct injection, AAV1 is more efficient than AAV2, and plasmid is inefficient in mediating gene transfer into the pig myocardium. AAV‐mediated VEGF gene transfer can also induce neovascular formation in the pig myocardium. Copyright