Hypoxic cardiocytes

At the end of the hypoxic period, viability of the cells were assessed by the Trypan Blue dye exclusion criteria. Fig. 15 demonstrated that the viability of hypoxic cells treated with IL were not significantly different form the viability of the normoxically cultured H9C2 cardiocytes. The viability of hypoxic cardiocytes treated with plain liposomes or IgG-liposomes was significantly lower than either those of immunoliposome treated hypoxic cells or normoxica cells. Since there was no difference in viability in cardiocytes treated... 

The sites of targeting of IL on hypoxic cardiocytes was visualized by fluorescent microscopy, using rhoda-mine labeled antimyosin immunoliposomes. Fig. 17 shows that only cells treated with antimyosin-rhoda-mine labeled IL were still confluent in the culture and that almost all cells were labeled with fluoresent liposomes. Those cells treated with rhodamine labeled PL showed extremely sparse number of cells still attached to the culture plates at 24 h of incubation, with essentially no or minimal fluorescence. Confocal microscopic examination of the cultures treated with rhodamine labeled IL showed that the cells still retained their morphology and shape with scattered fluorescent liposomes attached to the cell membranes (Fig. 18A). Those cells treated with rhodamine labeled PL were shrunken and only a few random cells showed some non-specific attachment of fluorescent PL (Fig. 18B). In this study, untreated hypoxic cells were all dead and since there was no fluorescent compounds added in them, no micrographs were obtained. 

It is also very important, how efficacious this protective effect can last. Therefore, we undertook a study to determine whether antimyosin IL can protect severely injured cardiocytes cultured under hypoxic conditions for 1-5 days. The experiment was designed as already described, however, cells were kept under hypoxic conditions during different times. Cell viability was assessed by H-Thymidine ( H-T) uptake. Untreated hypoxic cardiocytes (HC), normoxic cardiocytes (NC), and cardiocytes treated with PL were used as controls. Survival of NC increased from 100%... 

To demonstrate that preservation of cell viability is due to fusion of IL with the cell membranes, IL were prepared with intraliposomal silver grains. The rationale was that the only way silver grains can enter the treated hypoxic cardiocytes is by fusion of the IL with the cell membrane that resulted in preservation of cardiocyte viability. If endocytosis of the IL is the process of internalization without fusion, then plain liposomes should also show internalization of the silver grains. To prepare such liposomes,silver nitrate was added in a buffer solution during liposome preparation by soni-cation. Liposomes, purified from the non-entrapped silver nitrate by dialysis were dialyzed in 0.12 M NaCl solution and exposed to light for 1 h that led to the formation of fine electron-dense precipitate of silver oxide inside liposomes. 

Khaw, B.A. Torchilin, V.P. Vural, I. Narula, J. Plug and seal prevention of hypoxic cardiocyte death by sealing membrane lesions with antimyosin-liposomes. Nature Med. 1995,1, 1195-1198. 

Fig. 15 Comparison of cell viability of H9C2 cardiocytes in culture by Trypan Blue dye uptake criteria. Viability of hypoxic (24 h) cells treated the IL were compared to PL or IgG-liposome. Positive control was the normoxic cultures and the negative control was hypoxic cells with no treatment. (From Ref. l)...

Fig. 17 Epifluorescent micrographs of 24 h hypoxic H9C2 cardiocytes treated with rhodamine labeled IL (B) or rhodamine labeled PL (A). Cells treated with IL were all viable however, they all showed attached rhidamine fluorescent indicating attachment of IL to the cells. (From Ref. l)...

Fig. 20 Transmission electronmicrographs of hypoxic H9C2 cardiocytes treated with silver grains impregnated IL or PL. Silver grains in seen intracellularly in IL treated cells arrows, arrow heads represents 1 gm. (From Ref.[ " l)...

Survival of NCs as assessed by 3HT uptake, increased from 100% (the mean 3HT uptake in control NC was assigned 100%) to approx 254% after 24 h normal cell replication, whereas virtually no survival of HCs was registered after the same time period. Plain liposomes added to HC provided some protection, as evidenced by a decrease in viability after 24 h (approx 78%), of to approx 4% after 2 d of hypoxia, and essentially 0% after 3 d or more. When hypoxic cells were treated with IL, not only did IL confer protection, but it also permitted cell replication. This is evidenced by the similarity in the increase of 3HT uptake after 24 h of incubation between NCs (254%) and IL-treated HC (225%). After 48 h or more of hypoxia, replication in IL-treated HCs was decreased, but the viability of cells was maintained on the level of approx 90, 48, 15, and 7.8% after 2, 3, 4, and 5 d of hypoxia, respectively. These data show that the protective effect imposed by IL on hypoxic cells leads to a long-term preservation of cardiocyte viability, which might be especially important from a practical point of view for long-term preservation of myocardial viability. 

---