SUMMARY Chronic hypoxia in the presence of high glucose leads to progressive acidosis of cardiac myocytes in culture. The condition parallels myocardial ischemia in vivo, where ischemic tissue becomes rapidly hypoxic and acidotic. Cardiac myocytes are resistant to chronic hypoxia at neutral pH but undergo extensive death when the extracellular pH (pH[o]) drops below 6.5. A microarray analysis of 20 000 genes (cDNAs and expressed sequence tags)screened with cDNAs from aerobic and hypoxic cardiac myocytes identified>100 genes that were induced by >2-fold and ∼20 genes that were induced by >5-fold. One of the most strongly induced transcripts was identified as the gene encoding the pro-apoptotic Bcl-2 family member BNIP3. Northern and western blot analyses confirmed that BNIP3 was induced by 12-fold(mRNA) and 6-fold (protein) during 24 h of hypoxia. BNIP3 protein, but not the mRNA, accumulated 3.5-fold more rapidly under hypoxia–acidosis. Cell fractionation experiments indicated that BNIP3 was loosely bound to mitochondria under conditions of neutral hypoxia but was translocated into the membrane when the myocytes were acidotic. Translocation of BNIP3 coincided with opening of the mitochondrial permeability pore (MPTP). Paradoxically,mitochondrial pore opening did not promote caspase activation, and broad-range caspase inhibitors do not block this cell death pathway. The pathway was blocked by antisense BNIP3 oligonucleotides and MPTP inhibitors. Therefore,cardiac myocyte death during hypoxia–acidosis involves two distinct steps: (1) hypoxia activates transcription of the death-promoting BNIP3 gene through a hypoxia-inducible factor-1 (HIF-1) site in the promoter and (2) acidosis activates BNIP3 by promoting membrane translocation. This is an atypical programmed death pathway involving a combination of the features of apoptosis and necrosis. In this article, we will review the evidence for this unique pathway of cell death and discuss its relevance to ischemic heart disease. The article also contains new evidence that chronic hypoxia at neutral pH does not promote apoptosis or activate caspases in neonatal cardiac myocytes.

The localized delivery of exogenous, angiogenic growth factors has become a promising alternative treatment of peripheral artery disease (PAD) and critical limb ischemia. In the present study, we describe the development of a novel controlled release vehicle to promote angiogenesis in a murine critical limb ischemic model. Ionic, gelatin-based hydrogels were prepared by the carbodiimide-mediated amidation reaction between the carboxyl groups of gelatin or poly-L-glutamic acid molecules and the amine groups of poly-L-lysine or gelatin molecules, respectively. The degree of swelling of the synthesized hydrogels was assessed as a function of EDC/NHS ratios and the pH of the equilibrating medium, while the release kinetic profile of basic fibroblast growth factor (FGF-2) was evaluated in human fibroblast cultures. The degree of swelling (DS) decreased from 26.5+/-1.7 to 18.5+/-2.4 as the EDC concentration varied from 0.75 to 2.5 mg/ml. Eighty percent of the FGF-2 was released at controlled rates from gelatin-polylysine (gelatin-PLL) and gelatin-polyglutamic acid (gelatin-PLG) hydrogel scaffolds over a period of 28 days. Cell adhesion studies revealed that the negatively charged surface of the gelatin-PLG hydrogels exhibited superior adhesion capabilities in comparison to gelatin-PLL and control gelatin surfaces. Laser Doppler perfusion imaging as well as CD31(+) capillary immunostaining demonstrated that the controlled release of FGF-2 from ionic gelatin-based hydrogels is superior in promoting angiogenesis in comparison to the bolus administration of the growth factor. Over 4 weeks, FGF-2 releasing gelatin-PLG hydrogels exhibited marked reperfusion with a Doppler ratio of 0.889 (+/-0.04) which was 69.3% higher than in the control groups.

A single dose of a gene therapy vector carrying an inducible metalloproteinase human gene can both protect against the IOP increase produced by corticosteroid instillation in the sheep model and quickly reverse the IOP increase previously elicited by the corticosteroid. These results are a first step toward a treatment of steroid-glaucoma with inducible overexpression of extracellular matrix modulator genes.

Tubulysins are highly toxic tubulin-targeting agents with a narrow therapeutic window that are interesting for application in antibody-drug conjugates (ADC). For full control over drug-antibody ratio (DAR) and the effect thereof on pharmacokinetics and tumor targeting, a dual-labeling approach was developed, wherein the drug, tubulysin variants, and the antibody, the anti-HER2 monoclonal antibody (mAb) trastuzumab, are radiolabeled.131 I-radioiodination of two synthetic tubulysin A analogues, the less potent TUB-OH (IC 50 > 100 nmol/L) and the potent TUB-OMOM (IC 50 , $1 nmol/L), and their direct covalent conjugation to 89 Zrtrastuzumab were established. Radioiodination of tubulysins was 92% to 98% efficient and conversion to N-hydroxysuccinimide (NHS) esters more than 99%; esters were isolated in an overall yield of 68% AE 5% with radiochemical purity of more than 99.5%. Conjugation of 131 I-tubulysin-NHS esters to 89 Zr-trastuzumab was 45% to 55% efficient, resulting in ADCs with 96% to 98% radiochemical purity after size-exclusion chromatography. ADCs were evaluated for their tumor-targeting potential and antitumor effects in nude mice with tumors that were sensitive or resistant to trastuzumab, using ado-trastuzumab emtansine as a reference. ADCs appeared stable in vivo. An average DAR of 2 and 4 conferred pharmacokinetics and tumor-targeting behavior similar to parental trastuzumab. Efficacy studies using single-dose TUB-OMOM-trastuzumab (DAR 4) showed dose-dependent antitumor effects, including complete tumor eradications in trastuzumab-sensitive tumors in vivo. TUB-OMOM-trastuzumab (60 mg/kg) displayed efficacy similar to ado-trastuzumab emtansine (15 mg/kg) yet more effective than trastuzumab. Our findings illustrate the potential of synthetic tubulysins in ADCs for cancer treatment. Cancer Res; 74(20); 5700-10. Ó2014 AACR.

The authors have developed a novel glucocorticoid-inducible adenovirus vector that overproduces MMP1 only in the presence of DEX. The availability of this vector sets up the foundation for the development of gene therapy drugs for the potential treatment of ocular hypertension in steroid-responsive patients.