Generation in human plasma of misfolded, aggregation-prone electronegative low density lipoprotein.

Human plasma contains small amounts of a low density lipoprotein in which apoprotein is misfolded. Originally identified and isolated by means of anion-exchange chromatography, this component was subsequently described as electronegative low density lipoprotein (LDL)(-), with increased concentrations associated with elevated cardiovascular disease risk. It has been recognized recently as the trigger of LDL amyloidogenesis, which produces aggregates similar to subendothelial droplets observed in vivo in early atherogenesis. Although LDL(-) has been produced in vitro through various manipulations, the mechanisms involved in its generation in vivo remain obscure. By using a more physiological model, we demonstrate spontaneous, sustained and noticeable production of LDL(-) during incubation of unprocessed human plasma at 37 degrees C. In addition to a higher fraction of amyloidogenic LDL(-), LDL purified from incubated plasma contains an increased level of lysophospholipids and free fatty acids; analysis of LDL lipids packing shows their loosening. As a result, during plasma incubation, lipid destabilization and protein misfolding take place, and aggregation-prone particles are generated. All these phenomena can be prevented by inhibiting calcium-dependent secretory phospholipases A2. Our plasma incubation model, without removal of reaction products, effectively shows a lipid-protein interplay in LDL, where lipid destabilization after lipolysis threatens the apoprotein's structure, which misfolds and becomes aggregation-prone.

Low density lipoprotein misfolding and amyloidogenesis.

In early atherogenesis, subendothelial retention of lipidic droplets is associated with an inflammatory response-to-injury, culminating in the formation of foam cells and plaque. Low density lipoprotein (LDL) is the main constituent of subendothelial lipidic droplets. The process is believed to occur following LDL modification. Searching for a modified LDL in plasma, electronegative LDL [LDL(-)] was identified and found to be associated with major risk biomarkers. The apoprotein in LDL(-) is misfolded, and we show here that this modification primes the aggregation of native LDL, conforming to the typical pattern of protein amyloidogenesis. After a lag phase, whose length depends on LDL(-) concentration, light scattering and atomic force microscopy reveal early exponential growth of intermediate globules, which evolve into fibrils. These globules are remarkably similar to subendothelial droplets in atheromatous lesions and different from those produced by oxidation or biochemical manipulation. During aggregation, ellipticity and tryptophan fluorescence measurements reveal a domino-style spread of apoprotein misfolding from LDL(-) to all of the LDL. Computational analysis of the apoprotein primary sequence predicts an unstable, aggregation-prone domain in the regulatory alpha2 region. Apoprotein misfolding well represents an LDL modification able to transform this cholesterol carrier into a trigger for a response-to-injury in the artery wall.

Recombinant human nerve growth factor with a marked activity in vitro and in vivo.

Recombinant human nerve growth factor (rhNGF) is regarded as the most promising therapy for neurodegeneration of the central and peripheral nervous systems as well as for several other pathological conditions involving the immune system. However, rhNGF is not commercially available as a drug. In this work, we provide data about the production on a laboratory scale of large amounts of a rhNGF that was shown to possess in vivo biochemical, morphological, and pharmacological effects that are comparable with the murine NGF (mNGF), with no apparent side effects, such as allodynia. Our rhNGF was produced by using conventional recombinant DNA technologies combined with a biotechnological approach for high-density culture of mammalian cells, which yielded a production of approximately 21.5 +/- 2.9 mg/liter recombinant protein. The rhNGF-producing cells were thoroughly characterized, and the purified rhNGF was shown to possess a specific activity comparable with that of the 2.5S mNGF by means of biochemical, immunological, and morphological in vitro studies. This work describes the production on a laboratory scale of high levels of a rhNGF with in vitro and, more important, in vivo biological activity equivalent to the native murine protein.

Effect of exogenous administration of nerve growth factor in the retina of rats with inherited retinitis pigmentosa.

NGF is implicated in retinal damage regression. To study whether this is a direct effect or an effect mediated by NGF on other endogenous biological mediators, we investigated the effect of exogenous administration of NGF in RCS rats affected by retinitis pigmentosa. We found that NGF administration exerts a rescue effect on photoreceptors in this animal model. NGF injection enhances brain-derived neurotrophic factor, beta-fibroblast growth factor, transforming growth factor-beta, vascular endothelial factor and neuropeptide-Y. This suggests that NGF has an effect on RCS rat retina, probably also through the stimulation of other biological mediators produced and released in the retina.