Membrane microparticles are submicron fragments of membrane shed into extracellular space from cells under conditions of stress/injury. They may be distinguished from other classes of extracellular vesicles (i.e. exosomes) on the basis of size, content and mechanism of formation. Microparticles are found in plasma and other biological fluids from healthy individuals and their levels are altered in various diseases, including diabetes, chronic kidney disease, pre-eclampsia and hypertension among others. Accordingly, they have been considered biomarkers of vascular injury and pro-thrombotic or pro-inflammatory conditions. In addition to this, emerging evidence suggests that microparticles are not simply a consequence of disease, but that they themselves may contribute to pathological processes. Thus microparticles appear to serve as both markers and mediators of pathology. The present review examines the evidence for microparticles as both biomarkers of, and contributors to, the progression of disease. Approaches for the detection of microparticles are summarized and novel concepts relating to the formation of microparticles and their biological effects are examined.

Cyclin-dependent kinases (CDKs) are commonly known to regulate cell proliferation. However, previous reports suggest that in cultured postmitotic neurons, activation of CDKs is a signal for death rather than cell division. We determined whether CDK activation occurs in mature adult neurons during focal stroke in vivo and whether this signal was required for neuronal death after reperfusion injury. Cdk4͞cyclin D1 levels and phosphorylation of its substrate retinoblastoma protein (pRb) increase after stroke. Deregulated levels of E2F1, a transcription factor regulated by pRb, are also observed. Administration of a CDK inhibitor blocks pRb phosphorylation and the increase in E2F1 levels and dramatically reduces neuronal death by 80%. These results indicate that CDKs are an important therapeutic target for the treatment of reperfusion injury after ischemia.T he mechanism by which stroke-induced neuronal death occurs is complex and is likely dependent upon the severity and duration of ischemic insult and an elaborate interplay between ischemic death initiators such as excitotoxicity, oxidative stress, DNA damage, and inflammatory responses (1-3). Neurons that survive the acute ischemic injury undergo a delayed cell death that exhibits some characteristics of apoptosis (1-3). This delayed cell death is dependent upon selected death-signaling elements such as caspases, poly(ADP-ribose) polymerase, and p53 (4). The identification of signaling molecules that control delayed neuronal death has led to the hope that some of these death-signaling elements may serve as useful therapeutic targets for the reduction of neuropathology and behavioral deficits associated with stroke injury. The mechanism by which stroke-evoked delayed death occurs, however, is not fully understood.The cell cycle is a highly coordinated process regulated by the appropriate and timely activation of cyclin-dependent kinases (CDKs) (5). Regulation of CDKs is complex and includes binding to their obligate cyclin partner, activating and inhibitory phosphorylation events, and endogenous inhibitors of CDK activity. Distinct CDKs regulate progressive phases of the cell cycle. Generally, it is thought that the Cdk4͞6͞cyclin D1 complex regulates the G 0 to G 1 , Cdk2͞cyclin E and Cdk3 control G 1 to S, and Cdk2͞cyclin A and Cdk1͞cyclin B control G 2 and M progressions. Although the downstream targets of CDKs are not fully characterized, one important substrate is the tumor suppresser retinoblastoma protein (pRb), which is phosphorylated by activated Cdk4͞6͞cyclin D complex (6). Once hyperphosphorylated, pRb is released from the transcription factor complex E2F͞DP, which then activates genes required for S phase transition (7-9).Paradoxically, increasing evidence suggests that CDKs may have functions beyond that of cell cycle regulation. Numerous reports indicate the requirement of CDK signals for death of cultured postmitotic neurons exposed to select death insults. For example, inappropriate cyclin B and cyclin D1 transcripts have been observed in neuronal P...

p53 is a transcriptional activator which has been implicated as a key regulator of neuronal cell death after acute injury. We have shown previously that p53-mediated neuronal cell death involves a Bax-dependent activation of caspase 3; however, the transcriptional targets involved in the regulation of this process have not been identified. In the present study, we demonstrate that p53 directly upregulates Apaf1 transcription as a critical step in the induction of neuronal cell death. Using DNA microarray analysis of total RNA isolated from neurons undergoing p53-induced apoptosis a 5–6-fold upregulation of Apaf1 mRNA was detected. Induction of neuronal cell death by camptothecin, a DNA-damaging agent that functions through a p53-dependent mechanism, resulted in increased Apaf1 mRNA in p53-positive, but not p53-deficient neurons. In both in vitro and in vivo neuronal cell death processes of p53-induced cell death, Apaf1 protein levels were increased. We addressed whether p53 directly regulates Apaf1 transcription via the two p53 consensus binding sites in the Apaf1 promoter. Electrophoretic mobility shift assays demonstrated p53–DNA binding activity at both p53 consensus binding sequences in extracts obtained from neurons undergoing p53-induced cell death, but not in healthy control cultures or when p53 or the p53 binding sites were inactivated by mutation. In transient transfections in a neuronal cell line with p53 and Apaf1 promoter–luciferase constructs, p53 directly activated the Apaf1 promoter via both p53 sites. The importance of Apaf1 as a p53 target gene in neuronal cell death was evaluated by examining p53-induced apoptotic pathways in primary cultures of Apaf1-deficient neurons. Neurons treated with camptothecin were significantly protected in the absence of Apaf1 relative to those derived from wild-type littermates. Together, these results demonstrate that Apaf1 is a key transcriptional target for p53 that plays a pivotal role in the regulation of apoptosis after neuronal injury.