Ronald C. Hamdy

Official Positions of the International Society for Clinical Densitometry

Abstract The International Society for Clinical Densitometry (ISCD) has convened two Position Development Conferences at which a panel of experts agreed on recommendations for performance and clinical applications of bone density testing. These recommendation were reviewed by the ISCD Board of Directors, and those approved by the board are now official positions of the ISCD. These include (1) indications for bone density testing, (2) reference databases for T-scores, (3) standards for performing central dual-energy X-ray absorptiometry (DXA) for diagnosis, (4) interpretation of peripheral bone density results, (5) diagnosis of osteoporosis in postmenopausal women, (6) diagnosis of osteoporosis in men, (7) diagnosis in premenopausal women, (8) diagnosis in children, (9) indications and interpretation for serial bone mass measurement, (10) technical standards for phantom scanning and calibration, (11) technical standards for cross-calibration of DXA systems, and (12) standards for reporting of bone density results including correct nomenclature and preferred number of decimal digits.

Upregulation of vascular endothelial growth factor by H2O2 in rat heart endothelial cells

Hydrogen peroxide (H2O2) is a reactive oxygen species generated by several metabolic pathways in mammalian cells. Endothelial cells are extremely susceptible to oxidative stress. H2O2 has been reported to increase the permeability in these cells. Using rat heart endothelial cell culture as a model system, we examined the effect of H2O2 on the gene expression of vascular endothelial growth factor (VEGF), a potent mitogen of endothelial cells and a vascular permeability factor. By Northern blot analysis we found that VEGF mRNA responded to H2O2 in a dose-and time-dependent manner. The induction was superinduced by cycloheximide and blocked by actinomycin D. N-Acetylcysteine, a synthetic antioxidant, was able to suppress the induction. H7, a protein kinase C inhibitor, could also block the induction. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors, AP-1 and NF-kappaB. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 4 h after H2O2 stimulation. Our results demonstrate that VEGF gene expression is upregulated by H2O2 in these endothelial cells.

Upregulation of vascular endothelial growth factor by angiotensin II in rat heart endothelial cells

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and a vascular permeability factor. In this study we found that the addition of angiotensin II (AII) to rat heart endothelial cells induced VEGF mRNA production. VEGF mRNA levels reached a plateau within 2 h after the addition of AII and decreased after 4 h. The induction was superinduced by cycloheximide and blocked by actinomycin D. Losartan, an AT1 receptor antagonist, abolished the induction of VEGF mRNA by AII, whereas PD 123319, an AT2 receptor antagonist, had no effect on VEGF mRNA induction. H7, a protein kinase C inhibitor, blocked the induction. RT-PCR experiments showed two mRNA species (VEGF 120 and VEGF 164) in these cells and both species were stimulated by AII. Transient transfection experiment showed that VEGF promoter activity was increased 2.2-fold upon AII stimulation. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors AP-1 and NF-kappa B. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 8 h after AII stimulation. Our results demonstrate for the first time that the upregulation of VEGF by AII may play a significant role in AII-induced hyperpermeability.

Necrostatin-1 protects against glutamate-induced glutathione depletion and caspase-independent cell death in HT-22 cells

Glutamate, a major excitatory neurotransmitter in the CNS, plays a critical role in neurological disorders such as stroke and Parkinson’s disease. Recent studies have suggested that glutamate excess can result in a form of cell death called glutamate‐induced oxytosis. In this study, we explore the protective effects of necrostatin‐1 (Nec‐1), an inhibitor of necroptosis, on glutamate‐induced oxytosis. We show that Nec‐1 inhibits glutamate‐induced oxytosis in HT‐22 cells through a mechanism that involves an increase in cellular glutathione (GSH) levels as well as a reduction in reactive oxygen species production. However, Nec‐1 had no protective effect on free radical‐induced cell death caused by hydrogen peroxide or menadione, which suggests that Nec‐1 has no antioxidant effects. Interestingly, the protective effect of Nec‐1 was still observed when cellular GSH was depleted by buthionine sulfoximine, a specific and irreversible inhibitor of glutamylcysteine synthetase. Our study further demonstrates that Nec‐1 significantly blocks the nuclear translocation of apoptosis‐inducing factor (a marker of caspase‐independent programmed cell death) and inhibits the integration of Bcl‐2/adenovirus E1B 19 kDa‐interacting protein 3 (a pro‐death member of the Bcl‐2 family) into the mitochondrial membrane. Taken together, these results demonstrate for the first time that Nec‐1 prevents glutamate‐induced oxytosis in HT‐22 cells through GSH related as well as apoptosis‐inducing factor and Bcl‐2/adenovirus E1B 19 kDa‐interacting protein 3‐related pathways.

Humanin Is a Novel Neuroprotective Agent Against Stroke

Background and Purpose— Humanin (HN) is a 24-amino acid peptide best known for its ability to protect neurons from damage caused by Alzheimer disease-related proteins. This study examines the neuroprotective effects of HNG (a potent form of HN) on focal cerebral ischemia/reperfusion injury in mice. Methods— Mice underwent middle cerebral artery occlusion for 75 minutes followed by 24-hour reperfusion. Mice were pretreated with 0.1 &mgr;g HNG (intracerebroventricularly) 30 minutes before ischemia; posttreated at 0, 2, 4, and 6 hours after ischemia; or pretreated with 1 &mgr;g HNG (intraperitoneally) 1 hour before ischemia. Neurological deficits and cerebral infarct volume were evaluated. Neuronal apoptosis and activated poly(ADP-ribose) polymerase expression were measured by TUNEL and Western blot analysis, respectively. Activated ERKs were examined by Western blot analysis. Results— Pretreatment with 0.1 &mgr;g HNG (intracerebroventricularly) 30 minutes before ischemia reduced cerebral infarct volume from 56.2±3.0% to 26.1±1.4% (P<0.01). HNG posttreatment after 4 hours of reperfusion reduced cerebral infarct volume to 45.6±2.6% (P<0.05). Pretreatment with 1 &mgr;g HNG (intraperitoneally) 1 hour before ischemia or posttreatment after 2 hours of reperfusion reduced cerebral infarct volume significantly. HNG also significantly improved neurological function and inhibited both neuronal apoptosis as well as poly(ADP-ribose) polymerase activation. A significant decrease of phospho-ERK was observed in mice treated with HNG, whereas phospho-JNK and phospho-p38 levels were not altered. Conclusions— Our results demonstrate that HNG protects against cerebral ischemia/reperfusion injury in mice. HNG offers neuroprotection in vivo at least in part by inhibiting ERK activation. These findings suggest a potential therapeutic role for HNG in the treatment of stroke.

Synergistic protective effects of humanin and necrostatin-1 on hypoxia and ischemia/reperfusion injury

Since several different pathways are involved in cerebral ischemia/reperfusion injury, combination therapy rather than monotherapy may be required for efficient neuroprotection. In this study, we examined the protective effects of an apoptosis inhibitor Gly(14)-humanin (HNG) and a necroptosis inhibitor necrostatin-1 (Nec-1) on hypoxia/ischemia/reperfusion injury. Cultured mouse primary cortical neurons were incubated with Nec-1, HNG or both in a hypoxia chamber for 60 min. Cell viability was determined by MTS assay at 24h after oxygen-glucose deprivation (OGD) treatment. Mice underwent middle cerebral artery occlusion for 75 min followed by 24h reperfusion. Mice were administered HNG and/or Nec-1 (i.c.v.) at 4h after reperfusion. Neurological deficits were evaluated and the cerebral infarct volume was determined by TTC staining. Nec-1 or HNG alone had protective effects on OGD-induced cell death. Combined treatment with Nec-1 and HNG resulted in more neuroprotection than Nec-1 or HNG alone. Treatment with HNG or Nec-1 reduced cerebral infarct volume from 59.3 ± 2.6% to 47.0 ± 2.3% and 47.1 ± 1.5%, respectively. Combined treatment with HNG and Nec-1 improved neurological scores and decreased infarct volume to 38.6 ± 1.5%. In summary, we demonstrated that the combination treatment of HNG and Nec-1 conferred synergistic neuroprotection on hypoxia/ischemia/reperfusion injury in vitro and in vivo. These findings provide a novel therapeutic strategy for the treatment of stroke by combining anti-apoptosis and anti-necroptosis therapy.

Neuroprotective effect of humanin on cerebral ischemia/reperfusion injury is mediated by a PI3K/Akt pathway

Humanin (HN) is an anti-apoptotic peptide that suppresses neuronal cell death induced by Alzheimers disease, prion protein fragments, and serum deprivation. Recently, we demonstrated that Gly14-HN (HNG), a variant of HN in which the 14th amino acid serine is replaced with glycine, can decrease apoptotic neuronal death and reduce infarct volume in a focal cerebral ischemia/reperfusion mouse model. In this study, we postulate that the mechanism of HNGs neuroprotective effect is mediated by the PI3K/Akt pathway. Oxygen-glucose deprivation (OGD) was performed in cultured mouse primary cortical neurons for 60 min. The effect of HNG and PI3K/Akt inhibitors on OGD-induced cell death was examined at 24 h after reperfusion. HNG increased cell viability after OGD in primary cortical neurons, whereas the PI3K/Akt inhibitors wortmannin and Akti-1/2 attenuated the protective effect of HNG. HNG rapidly increased Akt phosphorylation, an effect that was inhibited by wortmannin and Akti-1/2. Mouse brains were injected intraventricularly with HNG before being subjected to middle cerebral artery occlusion (MCAO). HNG treatment significantly elevated p-Akt levels after cerebral I/R injury and decreased infarct volume. The protective effect of HNG on infarct size was attenuated by wortmannin and Akti-1/2. Taken as a whole, these results suggest that PI3K/Akt activation mediates HNGs protective effect against hypoxia/ischemia reperfusion injury.

Antioxidant activity of 7,8-dihydroxyflavone provides neuroprotection against glutamate-induced toxicity

Glutamate, an excitatory neurotransmitter in the central nervous system, plays an important role in neurological disorders. Previous studies have shown that excess glutamate can cause oxidative stress in a hippocampal HT-22 cell line. 7,8-Dihydroxyflavone (7,8-DHF), a member of the flavonoid family, is a selective tyrosine kinase receptor B (TrkB) agonist that has neurotrophic effects in various neurological diseases such as stroke and Parkinsons disease. In this study, we found that there is no TrkB receptor in HT-22 cells. Despite this, our data demonstrate that 7,8-DHF still protects against glutamate-induced toxicity in HT-22 cells in a concentration-dependent manner, indicating that 7,8-DHF prevents cell death through other mechanisms rather than TrkB receptors in this cell model. We further show that 7,8-DHF increases cellular glutathione levels and reduces reactive oxygen species (ROS) production caused by glutamate in HT-22 cells. Finally, our data demonstrate that 7,8-DHF protects against hydrogen peroxide and menadione-induced cell death, suggesting that 7,8-DHF has an antioxidant effect. In summary, although 7,8-DHF is considered as a selective TrkB agonist, our results demonstrate that 7,8-DHF can still confer neuroprotection against glutamate-induced toxicity in HT-22 cells via its antioxidant activity.

Overexpression of CuZnSOD in coronary vascular cells attenuates myocardial ischemia/reperfusion injury

Superoxide dismutase scavenges oxygen radicals, which have been implicated in ischemia/reperfusion (I/R) injury in the heart. Our experiments were designed to study the effect of a moderate increase of copper/zinc superoxide dismutase (CuZnSOD) on myocardial I/R injury in TgN(SOD1)3Cje transgenic mice. A species of 0.8 kb human CuZnSOD mRNA was expressed, and a 273% increase in CuZnSOD activity was detected in the hearts of transgenic mice with no changes in the activities of other antioxidant enzymes. Furthermore, immunoblot analysis revealed no changes in the levels of HSP-70 or HSP-25 levels. Immunocytochemical study indicated that there was increased labeling of CuZnSOD in the cytosolic fractions of both endothelial cells and smooth muscle cells, but not in the myocytes of the hearts from transgenic mice. When these hearts were perfused as Langendorff preparations for 45 min after 35 min of global ischemia, the functional recovery of the hearts, expressed as heart rate x LVDP, was 48 +/- 3% in the transgenic hearts as compared to 30 +/- 5% in the nontransgenic hearts (p <.05). The improved cardiac function was accompanied by a significant reduction in lactate dehydrogenase release from the transgenic hearts. Our results demonstrate that overexpression of CuZnSOD in coronary vascular cells renders the heart more resistant to I/R injury.

Long-term effects of strokes on bone mass

The purpose of the study was to determine the long-term effects of muscle weakness secondary to strokes on the bone mineral content of the hemiparetic limb. Patients who had experienced single recent strokes were studied. The bone mineral content of each limb was measured by Dual Energy X-ray Absorptiometry using the region of interest analysis program. Muscle strength of each muscle group was ranked using the Oxford scale, and the mean was calculated for each limb. Bone and muscle parameters were measured within seven days after the stroke and repeated thereafter at monthly intervals for up to 6 mo. A repeated measures analysis of variance, Newman-Keuls pair-wise comparisons, and orthogonal contrasts were done for each parameter. Significance levels were set at P < 0.05. Sixteen patients were included in this study. Demineralization was more pronounced in the upper than lower limbs. Demineralization of bones on the paralyzed side started during the first month after the stroke and gradually progressed. By the fourth month, the bone mineral content decreased by a mean of 9.3% (P = 0.01) and 3.7% (P = 0.01) in the upper and lower limbs, respectively, for the 11 patients followed for 4 mo. In the patients we followed for more than 4 mo, there was no further significant mineral loss. No change in bone mineral content was observed in the healthy nonparetic limbs. In conclusion, after a stroke, bone demineralization occurs in the paralyzed side and reaches its maximum within 3 to 4 mo. Arms are affected more than legs.