X-Message-Number: 14126
Date: Thu, 20 Jul 2000 05:21:27 -0700 (PDT)
From: Doug Skrecky <>
Subject: apoptosis and cold preservation

Citations: 1-4
  Protection by vascular endothelial growth factor against sinusoidal
  endothelial damage and apoptosis induced by cold
  Transplantation.  69(1):141-7, 2000 Jan 15.
  BACKGROUND: It is well known that sinusoidal endothelial cell (SEC) damage
  during cold preservation of liver tissue is closely involved in early graft
  failure. The objective of this study was to investigate the involvement of
  apoptosis in the SEC damage induced by cold preservation and
  to demonstrate the protective effect of vascular endothelial growth factor
  (VEGF) on SEC injury, including apoptotic changes. METHODS: Isolated SECs and
  liver tissue of Wistar rats were cold-preserved in University of Wisconsin
  (UW) solution, and the protective effect of VEGF was then investigated.
  Isolated SECs were cultured for 24 hr, and divided into the following 3
  groups: Group A, in which the cells were cultured for an additional 27 hr,
  Group B, in which the cells were cold-preserved in UW solution for 3 hr, and
  then recultured for 24 hr, and Group C, in which 20 ng/ml of VEGF was added
  to both the culture medium and the UW solution of cells cultured according to
  the Group B protocol. Each group of SECs was morphologically examined using
  the phase contrast microscopic method and the transmission electron
  microscopic method (TEM), and quantitatively analyzed using the WST-1 assay.
  Rat livers were cold-preserved in UW solution and divided into the VEGF(+)
  group and the VEGF(-) group, depending on whether VEGF was added or not. Each
  group of livers were analyzed by scanning electron microscopic method (SEM)
  after 24 hr of preservation. The hyaluronic acid uptake rate (HUR) was also
  determined after 6 hr of preservation. After 24 hr of preservation and 6 hr
  of reperfusion, tissues were examined by TEM and by the terminal
  deoxynucleotidyl transferase d-uridine triphosphate nick end labeling (TUNEL)
  assay. RESULTS: The phase contrast microscopic method and the WST-1 assay
  showed a protective effect of VEGF against the injury to isolated SECs during
  cold preservation and subsequent reculturing. Apoptosis was
  detected immediately by TEM after isolation of SECs, and the number of
  apoptotic cells increased with the incubation time. This increase was
  accelerated after cold preservation. The scanning electron microscopic method
  and the hyaluronic acid uptake rate showed a protective effect of VEGF
  against SEC damage in the cold-preserved livers. In the liver tissue, the TEM
  and the TUNEL assay detected apoptosis of SECs only after
  cold preservation and subsequent reperfusion. VEGF suppressed the
  apoptosis of SECs induced by cold preservation in both
  isolated cells and liver tissue. CONCLUSIONS: We demonstrated that SEC damage
  in the cold preservation of liver tissue was caused mainly by
  apoptosis, which required subsequent reperfusion. Moreover,
  isolated SECs showed spontaneous occurrence of apoptotic changes during
  culture, and these changes were accelerated by the preceding cold
  preservation. This is the first report to demonstrate the apoptotic changes
  of SECs seen here were inhibited by VEGF.

  Calpain inhibition prevents sinusoidal endothelial cell
  apoptosis in the cold ischemic rat liver.
  Transplantation.  68(1):136-40, 1999 Jul 15.
  BACKGROUND: Cold preservation of the liver followed by reperfusion results in
  sinusoidal endothelial cell (SEC) apoptosis. Calpain-like
  activity is dramatically increased during reperfusion and inhibition of
  calpains results in lower graft injury and longer survival. Recently,
  calpains have been implicated in inducing apoptosis. Our aim
  was to determine the effect of calpain inhibition on SEC
  apoptosis. METHODS: Livers were stored in the University of
  Wisconsin solution for 24 hr (survival conditions) and 40 hr (nonsurvival
  conditions) and ex vivo reperfused for 1 hr at 37 degrees C. Calpain-like
  activity was inhibited in some experiments using an i.p. injection of a
  selective inhibitor 2 hr before explantation. Apoptosis was
  quantified using the terminal deoxynucleotidyl trans. ferase-mediated dUTP
  nick end-labeling assay. Cross-inhibition by the inhibitor was determined for
  caspases 1 and 3. RESULTS: Apoptosis of exclusively the SEC
  was a key feature of reperfusion injury after both storage periods in
  University of Wisconsin solution after 1 hr normothermic reperfusion.
  Inhibition of calpain activity with Cbz-Val-Phe methyl ester resulted in a
  50% reduction of apoptotic SEC in the 40-hr preserved liver, and an almost
  complete abrogation of SEC apoptosis after 24 hr
  preservation. Only minimal cross-inhibition of caspases was determined at
  high concentrations in vitro by the calpain inhibitor. CONCLUSION:
  Apoptosis of exclusively SEC is a key feature of reperfusion
  injury partially mediated through calpain-dependent processes. Calpain
  inhibition reduces the number of apoptotic SEC. Based on these data and our
  previous work, calpain inhibition may prove to be useful in clinical

  Allograft heart valves: the role of apoptosis-mediated cell
  Journal of Thoracic & Cardiovascular Surgery.  117(3):454-62, 1999 Mar.
  OBJECTIVE: The purpose of this study was to determine whether
  apoptosis of endothelial and connective tissue cells is
  responsible for the loss of cellularity observed in implanted aortic
  allograft valves. METHODS: Fresh (n = 6) and cryopreserved (n = 4) aortic
  allograft valves were retrieved at 2 days to 20 weeks after implantation in
  an ovine model. Sections of these valves were studied with the use of
  histologic and electron microscopic methods, nick end-labeling and dual
  immunostaining for factor VIII-related antigen and proliferating cell nuclear
  antigen, followed by counterstaining for DNA and laser scanning confocal
  fluorescence microscopic observation. RESULTS: The endothelial cells and cusp
  connective tissue cells of implanted valvular allografts showed loss of
  proliferating cell nuclear antigen (indicative of cessation of mitotic
  activity) and evidence of apoptosis (nick end labeling). The
  latter was manifested by nuclear condensation and pyknosis, positive nick end
  labeling, and formation of intra- and extracellular apoptotic bodies derived
  from the fragmentation of apoptotic cells. These changes began to develop at
  2 days after implantation, peaking at 10 to 14 days, and became complete by
  20 weeks, at which time the valves had the typical acellular morphologic
  features of allografts implanted for long periods of time. CONCLUSIONS:
  Apoptosis occurs in endothelial cells and cuspal connective
  tissue cells of implanted allografts and appears to be a cause of their loss
  of cellularity. This apoptosis may be related to various
  factors, including immunologic and chemical injury, and hypoxia during valve
  processing and reperfusion injury at the time of implantation.

  Cell death during corneal storage at 4 degrees C.
  Investigative Ophthalmology & Visual Science.  40(12):2827-32, 1999 Nov.
  PURPOSE: To evaluate cell death in human donor corneas stored at 4 degrees C,
  to determine whether terminal deoxynucleotidyl transferase-mediated
  dUTP-fluorescein nick-end labeling (TUNEL) discriminates between
  apoptosis and necrosis in corneas stored at 4 degrees C.
  METHODS: Ten human corneas were stored in Optisol (Chiron Ophthalmics,
  Irvine, CA) at 4 degrees C for periods ranging from 0 to 21 days and then
  fixed for histologic examination. Central corneal sections from each cornea
  were examined by transmission electron microscopy (TEM) and by the TUNEL
  assay. Electron micrographs of at least 15 keratocytes each from the
  anterior, middle, and posterior stroma were examined by three masked
  observers who graded each cell as normal, apoptotic, or necrotic. Central
  sections from the same corneas were processed by the TUNEL assay and
  evaluated with a laser scanning confocal microscope to determine the
  percentage of apoptotic cells. RESULTS: By TEM, apoptosis
  occurred in 23% of the keratocytes and necrosis in 12%. By TUNEL assay,
  apoptosis occurred in 11% of the keratocytes, with the
  results in individual corneas being similar to the findings by TEM for
  apoptosis, rather than for necrosis. By TUNEL assay,
  apoptosis occurred in 13% of the epithelial cells and in 8%
  of the endothelial cells. The percentage of apoptotic cells and storage time
  correlated significantly for the epithelium, but not for the keratocytes or
  endothelium in this small sample. CONCLUSIONS: Both
  apoptosis and necrosis occur in cells during corneal storage
  at 4 degrees C, with apoptosis appearing to predominate. The
  TUNEL assay identifies cells undergoing apoptosis, but not
  necrosis, in corneal tissue. Inhibition of apoptosis in
  corneas stored at 4 degrees C may prolong acceptable storage times.

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