A prospective evaluation of the biochemical, metabolic, hormonal and structural bone changes associated with bortezomib response in multiple myeloma patients.

We prospectively evaluated the bone changes associated with proteasome inhibition using single agent bortezomib in relapsed or refractory myeloma patients. Ten patients received bortezomib 1.3 mg/m(2) per days 1, 4, 8 and 11 for three 21-day cycles, and 6 patients received 1 mg/m(2) per day with the same schedule. Bone architecture and metabolism changes were assessed by bone markers, micro-CT, bone histomorphometry, tetracycline labeling and serum parathormone levels. Bone parameter variations were compared by response to treatment. Microarchitectural changes were observed in all evaluable responsive patients. Bone alkaline phosphatase changes were associated with disease response (≥PR vs. others P=0.03 cycle 1, day 11) serum parathormone levels were also significantly increased (P=0.04 on days 11, 21, 33) in responding individuals. This study demonstrates that the myeloma control produced by proteasome inhibition is associated with bone changes and to a discrete pattern of hormonal variation.

Role of A kinase anchor proteins in the tissue-specific regulation of lipoprotein lipase.

Activation of protein kinase A by catecholamines inhibits lipoprotein lipase (LPL) activity through the elaboration of an RNA binding complex, which inhibits LPL translation by binding to the 3'-untranslated region of the LPL mRNA. To better define this process, we reconstituted the inhibitory RNA binding complex in vitro and demonstrated that the K homology (KH) domain of A kinase anchor protein (AKAP) 121/149 plays a vital role in the inhibition of LPL translation. Inhibition of LPL translation occurred in vitro only when the Calpha subunit, R subunit, and AKAP 149 were present. Using different glutathione-S-transferase fusion proteins of AKAP 149, sequences containing the KH domain were required for inhibition of LPL translation, and the inhibition of AKAP 121 expression in 3T3-F442A adipocytes with short interfering RNA resulted in loss of epinephrine-mediated translation inhibition. After epinephrine injection into mice, LPL activity was inhibited in white adipose tissue but not in brown adipose tissue (BAT) or muscle. LPL activity and synthetic rate were inhibited in vitro by the addition of epinephrine to 3T3-F442A adipocytes, but there was no effect in L6 muscle cells and cultures of brown adipocytes. Corresponding with these differences in LPL translation, AKAP 121 protein and mRNA were abundantly expressed in mouse white adipose tissue, but was either very low or undetectable in BAT and muscle. Thus, AKAP 121/149 contains a KH region that is essential to the translation inhibition of LPL in response to epinephrine. BAT and muscle do not express significant AKAP 121/149, and this likely explains some of the tissue-specific differences in LPL regulation.

Low density lipoproteins modulate collagen metabolism in fibroblasts.

High levels of low-density lipoprotein are associated with atherosclerosis, and myocardial and arterial remodeling. We postulated that low-density lipoprotein influences collagen synthesis and degradation in fibroblasts as a potential mechanism of tissue remodeling. Incubation of cultured human skin fibroblasts with low-density lipoproteins resulted in a time-dependent and dose-dependent increase in the secretion of matrix metalloproteinase activity measured by gelatin zymography. Western blot analysis showed a concomitant increase in matrix metalloproteinase-1 protein. Northern blot analysis demonstrated an increase in collagen I messenger RNA after treatment with low-density lipoprotein. The matrix metalloproteinase-1 secretory response of fibroblasts to low-density lipoprotein was attenuated by heparin, which inhibits low-density lipoprotein uptake through the low-density lipoprotein-receptor. These observations suggest that low-density lipoprotein has a regulatory effect on collagen metabolism in fibroblasts.

LOX-1 mediates oxidized low-density lipoprotein-induced expression of matrix metalloproteinases in human coronary artery endothelial cells.

BACKGROUND: Oxidized LDL (ox-LDL) accumulation in the atherosclerotic region may enhance plaque instability. Both accumulation of ox-LDL and expression of its lectin-like receptor, LOX-1, have been shown in atherosclerotic regions. This study was designed to examine the role of LOX-1 in the modulation of metalloproteinases (MMP-1 and MMP-3) in human coronary artery endothelial cells (HCAECs). METHODS AND RESULTS: HCAECs were incubated with ox-LDL (10 to 80 micro g/mL) for 1 to 24 hours. Ox-LDL increased the expression of MMP-1 (collagenase) and MMP-3 (stromelysin-1) in a concentration- and time-dependent manner. Ox-LDL also increased collagenase activity. Ox-LDL did not significantly affect the expression of tissue inhibitors of metalloproteinases. Native LDL had no effect on the expression of MMPs. The effects of ox-LDL were mediated by its endothelial receptor, LOX-1, because pretreatment of HCAECs with a blocking antibody to LOX-1 (JTX92, 10 micro g/mL) prevented the expression of MMPs in response to ox-LDL (P<0.01). In parallel experiments, ox-LDL caused the activation of protein kinase C (PKC), which was inhibited by LOX-1 antibody. The PKC-beta isoform played a critical role in the expression of MMPs, because the PKC-beta inhibitor hispidin reduced ox-LDL-induced activation of PKC and the expression of MMPs. Other PKC subunits (alpha, gamma, and epsilon) did not affect the expression of MMPs. CONCLUSIONS: These findings indicate that ox-LDL, via LOX-1 activation, modulates the expression and activity of MMPs in HCAECs. In this process, activation of the PKC-beta subunit plays an important signaling role.

Aspirin inhibits human coronary artery endothelial cell proliferation by upregulation of p53.

Aspirin (acetylsalicylic acid, ASA) is effective in the primary and secondary prevention of vascular events. This effect is mediated in large part by platelet inhibition; however, non-platelet-mediated effects may also be relevant in the overall efficacy of ASA. We determined the effect of ASA on the synthesis of DNA and total proteins in cultured human coronary endothelial cells (HCAECs). Fourth generation HCAECs were cultured and treated with ASA and rate of synthesis of DNA and total proteins was determined by incorporation of [3H]thymidine and [3H]proline, respectively. ASA inhibited DNA synthesis by 50% at a concentration of 1mM and protein synthesis by 50% at a concentration of 2mM. The inhibitory effect of ASA was observed as early as 2h after treatment of HCAECs. The inhibition of DNA and protein synthesis could be reversed within 24h after removal of the drug from the culture medium. Indomethacin also inhibited DNA and protein synthesis. Western blot analysis revealed that the expression of p53 protein was increased after treatment of the cells with ASA. These observations indicate that ASA decreases endothelial cell proliferation through cell cycle arrest mediated by enhanced p53 expression. Arrest of endothelial proliferation and activation may be an important mechanism of the beneficial effect of ASA in acute coronary syndromes.