Intrathecal drug delivery in the era of nanomedicine.

Administration of substances directly into the cerebrospinal fluid (CSF) that surrounds the brain and spinal cord is one approach that can circumvent the blood-brain barrier to enable drug delivery to the central nervous system (CNS). However, molecules that have been administered by intrathecal injection, which includes intraventricular, intracisternal, or lumbar locations, encounter new barriers within the subarachnoid space. These barriers include relatively high rates of turnover as CSF clears and potentially inadequate delivery to tissue or cellular targets. Nanomedicine could offer a solution. In contrast to the fate of freely administered drugs, nanomedicine systems can navigate the subarachnoid space to sustain delivery of therapeutic molecules, genes, and imaging agents within the CNS. Some evidence suggests that certain nanomedicine agents can reach the parenchyma following intrathecal administration. Here, we will address the preclinical and clinical use of intrathecal nanomedicine, including nanoparticles, microparticles, dendrimers, micelles, liposomes, polyplexes, and other colloidalal materials that function to alter the distribution of molecules in tissue. Our review forms a foundational understanding of drug delivery to the CSF that can be built upon to better engineer nanomedicine for intrathecal treatment of disease.

Osteochondral avulsion fracture of the posteromedial tibial plateau.

Posteromedial tibial plateau avulsion fracture caused by semimembranosus muscle is not easy to detect by X-ray. The literature regarding this issue is poor, also mechanism is extensively disputable. This lesion was often connected to an anterior cruciate ligament (ACL) rupture and medial meniscal horn lesion. In this work, we described a posteromedial tibial plateau avulsion fracture at the semimembranosus insertion. In particular, we referred to the surgical treatment of those transversal osteochondral fractures.

Fate of nanoparticles in the central nervous system after intrathecal injection in healthy mice.

Cerebrospinal fluid (CSF) is produced in the cerebral ventricles and circulates within the subarachnoid space (SAS) of the brain and spinal cord, where it exchanges with interstitial fluid of the parenchyma. The access of CSF to the entire central nervous system (CNS) makes it an attractive medium for drug delivery. However, few intrathecal (IT) therapies have reached the clinic due, in part, to limited distribution and rapid clearance. Given the success of nanoparticle (NP) carriers in prolonging circulation and improving delivery of systemically administered agents, we sought to evaluate the distribution of IT injected NPs within the CNS. We administered fluorescent, 100 nm PEGylated-NPs into the cisterna magna of healthy mice and studied their distribution along the brain and spinal cord. Our data demonstrate that NPs are capable of distributing rapidly through the SAS along the entire neuraxis with reproducible, anatomically defined patterns of delivery. NPs were well retained within the leptomeninges for over 3 weeks, showing preference for ventral surfaces and minimal penetration into the CNS parenchyma. Clearance of NPs occurred across the cribriform plate into the nasal mucosa, with a small fraction of NPs localizing with nerve roots exiting the spinal column. Larger 10 µm particles were also capable of moving through the SAS but did not achieve as widespread distribution. These studies demonstrate the ability of NPs to achieve widespread delivery along the neuraxis and highlight IT administration as a potentially significant route of administration for delivery of nanomedicine to the subarachnoid space.

Hyaluronic acid-laminin hydrogels increase neural stem cell transplant retention and migratory response to SDF-1α.

The chemokine SDF-1α plays a critical role in mediating stem cell response to injury and disease and has specifically been shown to mobilize neural progenitor/stem cells (NPSCs) towards sites of neural injury. Current neural transplant paradigms within the brain suffer from low rates of retention and engraftment after injury. Therefore, increasing transplant sensitivity to injury-induced SDF-1α represents a method for increasing neural transplant efficacy. Previously, we have reported on a hyaluronic acid-laminin based hydrogel (HA-Lm gel) that increases NPSC expression of SDF-1α receptor, CXCR4, and subsequently, NPSC chemotactic migration towards a source of SDF-1α in vitro. The study presented here investigates the capacity of the HA-Lm gel to promote NPSC response to exogenous SDF-1α in vivo. We observed the HA-Lm gel to significantly increase NPSC transplant retention and migration in response to SDF-1α in a manner critically dependent on signaling via the SDF-1α-CXCR4 axis. This work lays the foundation for development of a more effective cell therapy for neural injury, but also has broader implications in the fields of tissue engineering and regenerative medicine given the essential roles of SDF-1α across injury and disease states.

Enhancing neural stem cell response to SDF-1α gradients through hyaluronic acid-laminin hydrogels.

Traumatic brain injury (TBI) initiates an expansive biochemical insult that is largely responsible for the long-term dysfunction associated with TBI; however, current clinical treatments fall short of addressing these underlying sequelae. Pre-clinical investigations have used stem cell transplantation with moderate success, but are plagued by staggeringly low survival and engraftment rates (2-4%). As such, providing cell transplants with the means to better dynamically respond to injury-related signals within the transplant microenvironment may afford improved transplantation survival and engraftment rates. The chemokine stromal cell-derived factor-1α (SDF-1α) is a potent chemotactic signal that is readily present after TBI. In this study, we sought to develop a transplantation vehicle to ultimately enhance the responsiveness of neural transplants to injury-induced SDF-1α. Specifically, we hypothesize that a hyaluronic acid (HA) and laminin (Lm) hydrogel would promote 1. upregulated expression of the SDF-1α receptor CXCR4 in neural progenitor/stem cells (NPSCs) and 2. enhanced NPSC migration in response to SDF-1α gradients. We demonstrated successful development of a HA-Lm hydrogel and utilized standard protein and cellular assays to probe NPSC CXCR4 expression and NPSC chemotactic migration. The findings demonstrated that NPSCs significantly increased CXCR4 expression after 48 h of culture on the HA-Lm gel in a manner critically dependent on both HA and laminin. Moreover, the HA-Lm hydrogel significantly increased NPSC chemotactic migration in response to SDF-1α at 48 h, an effect that was critically dependent on HA, laminin and the SDF-1α gradient. Therefore, this hydrogel serves to 1. prime NPSCs for the injury microenvironment and 2. provide the appropriate infrastructure to support migration into the surrounding tissue, equipping cells with the tools to more effectively respond to the injury microenvironment.

Selective GPER activation decreases proliferation and activates apoptosis in tumor Leydig cells.

We have previously shown that estrogens binding to estrogen receptor (ER) α increase proliferation of Leydig tumor cells. Estrogens can also bind to G protein-coupled ER (GPER) and activation of this receptor can either increase or decrease cell proliferation of several tumor types. The aim of this study was to investigate GPER expression in R2C rat tumor Leydig cells, evaluate effects of its activation on Leydig tumor cell proliferation and define the molecular mechanisms triggered in response to its activation. R2C cells express GPER and its activation, using the specific ligand G-1, is associated with decreased cell proliferation and initiation of apoptosis. Apoptosis after G-1 treatment was asserted by appearance of DNA condensation and fragmentation, decrease in Bcl-2 and increase in Bax expression, cytochrome c release, caspase and poly (ADP-ribose) polymerase-1 (PARP-1) activation. These effects were dependent on GPER activation because after silencing of the gene, using a specific small interfering RNA, cyt c release, PARP-1 activation and decrease in cell proliferation were abrogated. These events required a rapid, however, sustained extracellular regulated kinase 1/2 activation. G-1 was able to decrease the growth of R2C xenograft tumors in CD1 nude mice while increasing the number of apoptotic cells. In addition, in vivo administration of G-1 to male CD1 mice did not cause any alteration in testicular morphology, while cisplatin, the cytotoxic drug currently used for the therapy of Leydig tumors, severely damaged testicular structure, an event associated with infertility in cisplatin-treated patients. These observations indicate that GPER targeting for the therapy of Leydig cell tumor may represent a good alternative to cisplatin to preserve fertility in Leydig tumor patients.

DAX-1, as an androgen-target gene, inhibits aromatase expression: a novel mechanism blocking estrogen-dependent breast cancer cell proliferation.

Sexual hormones, estrogens and androgens, determine biological response in a tissue- and gender-specific manner and have a pivotal role in endocrine-mediated tumorigenesis. In situ estrogen production by aromatase is a critical determinant for breast cancer growth and progression. On the contrary, clinical and in vitro studies indicate that androgens have a protective role in mammary carcinogenesis. Here, we demonstrated, in hormone-dependent breast cancer cells, the existence of a functional interplay between the androgen receptor (AR), the orphan nuclear receptor DAX-1 and the aromatase enzyme involved in the inhibition of the estrogen-dependent breast cancer cell proliferation exerted by androgen signaling. Indeed, our results revealed, in MCF-7 cells, that ligand-activated AR induces the expression of the orphan nuclear receptor DAX-1 by direct binding to a newly identified androgen-response-element within the DAX-1 proximal promoter. In turn, androgen-induced DAX-1 is recruited, in association with the corepressor N-CoR, within the SF-1/LRH-1 containing region of the aromatase promoter, thereby repressing aromatase expression and activity. In elucidating a novel mechanism by which androgens, through DAX-1, inhibit aromatase expression in breast cancer cell lines, these findings reinforce the theory of androgen- opposing estrogen-action, opening new avenues for therapeutic intervention in estrogen-dependent breast tumors.

Gper and ESRs are expressed in rat round spermatids and mediate oestrogen-dependent rapid pathways modulating expression of cyclin B1 and Bax.

Spermatogenesis is a precisely controlled and timed process, comprising mitotic divisions of spermatogonia, meiotic divisions of spermatocytes, maturation and differentiation of haploid spermatids giving rise to spermatozoa. It is well known that the maintenance of spermatogenesis is controlled by gonadotrophins and testosterone, the effects of which are modulated by a complex network of locally produced factors, including oestrogens. However, it remains uncertain whether oestrogens are able to activate rapid signalling pathways directly in male germ cells. Classically, oestrogens act by binding to oestrogen receptors (ESRs) 1 and 2. Recently, it has been demonstrated that rapid oestrogen action can also be mediated by the G-protein-coupled oestrogen receptor 1 (Gper). The aim of the present study was to investigate ESRs and Gper expression in primary cultures of adult rat round spermatids (RS) and define if oestradiol (E2) is able to activate, through these receptors, pathways involved in the regulation of genes controlling rat RS apoptosis and/or maturation. In this study, we demonstrated that rat RS express ESR1, ESR2 and Gper. Short-time treatment of RS with E2, the selective Gper agonist G1 and the selective ESR1 and ERß agonists, 4,4',4"-(4-propyl-[1H]pyrazole-1,3,5-triyl) trisphenol (PPT) and 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), respectively, determined activation of Extra-cellular signal-regulated kinase (ERK1/2) through the involvement of epidermal growth factor receptor transactivation. In addition, we investigated the effects of ESRs and Gper pathway activation on factors involved in RS maturation. Expression of cyclin B1 mRNA was downregulated by E2, G1 and PPT, but not by DPN. A concomitant and inverse regulation of the pro-apoptotic factor Bax mRNA expression was observed in the same conditions, with DPN being the only one determining an increase in this factor expression. Collectively, these data demonstrate that E2 activates, through ESRs and Gper, pathways involved in the regulation of genes controlling rat RS apoptosis and differentiation such as cyclin B1 and Bax.

Antiestrogens upregulate estrogen receptor beta expression and inhibit adrenocortical H295R cell proliferation.

The molecular mechanisms involved in adrenocortical tumorigenesis are still not completely understood. In this study, using the H295R cell line as a model system, we investigated the role of estrogens and estrogen receptor (ER) alpha and ER beta in the growth regulation of adrenocortical tumors. We demonstrated that H295R cells are able to convert androgens to estrogens by a constitutive expression of active cytochrome P450 aromatase protein and express ER beta to a greater extent than ER alpha. Moreover, physiological concentrations of 17beta-estradiol (E2) determined an increase of thymidine incorporation, suggesting the presence of an autocrine mechanism in maintaining H295R cell proliferation. Evaluating the response to ER antagonists like 4-hydroxytamoxifen (OHT) and ICI 182 780 (ICI), we observed an up-regulation of ER beta and a dose-dependent inhibition of H295R cell proliferation. Whereas ICI determined the growth arrest of H295R cells, OHT induced morphological changes that were characteristic of apoptosis. According to the above-mentioned observations, OHT but not ICI clearly induced a marked expression of FasL and the cleavage of both caspase-8 and caspase-3. Interestingly, the apoptotic effects of OHT in H295R cells may be consequent to the enhanced levels of ER beta which stimulate the expression of FasL interacting with activating protein (AP)-1 sites located within its promoter sequence. In conclusion, we have demonstrated that H295R cells are able to transform androgens to estrogens that activate an autocrine mechanism, mediated by their own receptors, and contribute to regulate the proliferation of these cells. Moreover, this study points towards a role for ER beta as an important mediator of the repressive effects exerted by antiestrogens on H295R cells; however, further studies are needed to clarify its role in the control of adrenocortical cell proliferation and on the potential benefits of antiestrogens for treatment of adrenocortical cancer.

Inhibition of Src tyrosine kinase stimulates adrenal androgen production.

A unique characteristic of the primate adrenal is the ability to produce 19-carbon steroids, often called the adrenal androgens. Although it is clear that the major human adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEA-S), are produced almost solely in the adrenal reticularis, the mechanisms regulating production are poorly understood. Herein, we tested the hypothesis that the Src family of tyrosine kinases are involved in the regulation of adrenal androgen production. The NCI-H295R human adrenal cell line and primary human adrenal cells in culture were used to study adrenal androgen production and expression of enzymes involved in steroidogenesis. To examine the role of Src tyrosine kinase, cells were treated with PP2, a specific Src inhibitor. Alternatively, adrenal cells were transfected with an expression vector containing a dominant-negative form of Src. PP2 treatment inhibited basal cortisol production while significantly increasing the production of DHEA and DHEA-S (together referred to as DHEA(S)) in both adrenal cell models. The effect of PP2 on steroidogenesis occurred along with a rapid induction of steroidogenic acute regulatory (StAR) protein synthesis as revealed by Western analysis. Treatment with PP2 also increased mRNA levels for StAR, and cholesterol side-chain cleavage (CYP11A) and 17alpha-hydroxylase/17,20-lyase (CYP17) enzymes. Treatment of adrenal cells with the cAMP agonist dibutyryladenosine cyclic monophosphate (dbcAMP), stimulated the production of cortisol and DHEA(S). However, treatment of adrenal cells with a combination of PP2 and dbcAMP enhanced the production of DHEA(S) while inhibiting cortisol production. During dbcAMP treatment PP2 was able to augment the expression of CYP17 and to inhibit the induction of 3beta-hydroxysteroid dehydrogenase type 2 (HSD3B2) levels. Increasing the CYP17 to HSD3B2 ratio is likely to promote the use of steroid precursors for the production of DHEA(S) and not for cortisol. Taken together these data suggest that the inhibition of Src tyrosine kinases causes adrenal cells to adopt a reticularis phenotype both by the production of DHEA(S) and by the steroidogenic enzymes expressed.

Liver receptor homologue-1 is expressed in human steroidogenic tissues and activates transcription of genes encoding steroidogenic enzymes.

In the current study we test the hypothesis that liver receptor homologue-1 (LRH; designated NR5A2) is involved in the regulation of steroid hormone production. The potential role of LRH was assessed by first examining expression in human steroidogenic tissues and second by examining effects on transcription of genes encoding enzymes involved in steroidogenesis. LRH is closely related to steroidogenic factor 1 (SF1; designated NR5A1), which is expressed in most steroidogenic tissues and regulates expression of several steroid-metabolizing enzymes. LRH transcripts were expressed at high levels in the human ovary and testis. Adrenal and placenta expressed much lower levels of LRH than either ovary or liver. To examine the effects of LRH on steroidogenic capacity we used reporter constructs prepared with the 5'-flanking region of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage (CYP11A1), 3beta hydroxysteroid dehydrogenase type II (HSD3B2), 17alpha hydroxylase, 17,20 lyase (CYP17), 11beta hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2). Co-transfection of these reporter constructs with LRH expression vector demonstrated that like SF1, LRH enhanced reporter activity driven by flanking DNA from StAR, CYP11A1, CYP17, HSD3B2, and CYP11B1. Reporter constructs driven by CYP11A1 and CYP17 were increased the most by co-transfection with LRH and SF1. Of the promoters examined only HSD3B2 was more sensitive to LRH than SF1. The high level of ovarian and testicular LRH expression make it likely that LRH plays an important role in the regulation of gonadal function.

Effects of tri-iodothyronine on alternative splicing events in the coding region of cytochrome P450 aromatase in immature rat Sertoli cells.

Transient postnatal hypothyroidism in male rats induces a prolonged proliferation of immature Sertoli cells. This change in Sertoli cell replication at young ages is coincident with enhanced and prolonged aromatase activity that leads to a marked increase in the conversion of androgens into estrogens. Both events are drastically inhibited by tri-iodothyronine (T(3)) replacement either in vivo or in vitro. This study, after the immunolocalization of aromatase in cultured rat Sertoli cells, examined the effects elicited by T(3) on this enzyme, by simultaneously investigating three functional levels of aromatase: mRNA expression, protein content, and enzymatic activity. The immunolocalization of cytochrome P450 aromatase (P450 arom) was shown in the cytoplasm of cultured Sertoli cells from 15- and 21-day-old rats. Western blot analysis revealed an enhancement of aromatase protein content upon stimulation with N(6),2'-O-dibutyryladenosine-3':5'-cyclic monophosphate ((Bu)(2)cAMP) that was clearly down-regulated by T(3). The presence of a functional P450 arom protein in purified Sertoli cells was confirmed by the measurement of [(3)H]H(2)O released after incubation with [1 beta-(3)H]androst-4-ene-3,17-dione. With 100 nM T3, a decrease in both P450 arom mRNA levels and aromatase activity was observed. The aromatase enzymatic activity was strongly stimulated by (Bu)(2)cAMP and markedly down-regulated by T(3). In contrast, the strong increase in aromatase mRNA upon (Bu)(2)cAMP stimulation was apparently unaffected by T(3) administration. This paper shows how the identification of an altered transcript induced by T(3) coding for putative truncated and inactive aromatase protein might explain such a decrease in aromatase activity in T(3)-treated cells. On the basis of these results, it is concluded that at least two mechanisms could be involved in the down-regulatory effect of T(3) on aromatase activity in prepuberal Sertoli cells. The first mechanism is linked to a possible direct modulatory role for T(3) in the regulation of the aromatase promoter, whilst the second one is represented by the induction of altered transcripts coding for truncated and inactive aromatase proteins.

Aromatase expression in prepuberal Sertoli cells: effect of thyroid hormone.

Aromatase activity has recently been assumed as a Sertoli cell functional maturation marker since it is maximally expressed in prepuberal age then it dramatically decreases at puberty and is virtually absent in adult age. Neonatal hypothyroidism is associated with a prolonged proliferation of Sertoli cells. This immature stage persists concomitantly with a dramatic enhancement of aromatase activity reversed by triiodothyronine (T3) either in vivo or in vitro administration. Therefore, in the present study, after immunolocalisation of aromatase in the cytoplasm of cultured Sertoli cells as well as in testis section, we investigate the regulatory effects of T3 in the same cells just at the age when aromatase activity is reported to be maximally expressed. In this aim, the effects of thyroid hormone have been evaluated in 2-weeks-old rats, in basal condition and upon stimulation with dibutyryl cyclic AMP [(Bu)(2)cAMP] by simultaneously analysing three functional levels of aromatase, mRNA expression; protein content; enzymatic activity. Western-blot analysis of Sertoli cell extracts revealed a protein, which co-migrated with a 55 kDa protein detected in human placenta used a positive control. The presence of a functional P450 aromatase protein in purified Sertoli cells was confirmed by the measurement [3H]H(2)O released after incubation with [1beta-(3)H]androst-4-3,17-dione. At the dose used, T3 down-regulates basal aromatase activity, while aromatase mRNA expression was apparently not inhibited. It is noteworthy that aromatase content pattern evaluated by Western blot analysis did not tightly parallel the aromatase activity pattern which clearly displays the inhibitory effects of T3, in basal condition ad upon (Bu)(2)cAMP stimulation, simulating FSH stimulation. The detection of mRNA altered transcript coding for putative protein lacking both aromatic and heme-binding regions upon T3 treatment and unable to convert androgens into estrogens, provides a reasonable explanation for the observed discrepancies between aromatase protein pattern, P450arom mRNA levels and aromatase activity. The authors conclude that although the altered transcript induced by prolonged exposure to T3 is a mechanism by which T3 may down regulate aromatase activity, it cannot be ruled out a direct effect of this hormone at the transcription levels since a recognisable emisite for potential TR(s) binding is located in the promoter region of aromatase gene. Thus a further investigation on T3 modulator role on aromatase gene promoter should be pursued even utilising higher doses of T3.

A role for src tyrosine kinase in regulating adrenal aldosterone production.

Adrenal aldosterone synthesis is influenced by a variety of factors. The major physiological regulators of aldosterone production are angiotensin II (Ang IotaIota) and potassium (K(+)). Ang IotaIota stimulates aldosterone production through the activation of multiple intracellular signaling pathways. It has recently been demonstrated that Ang IotaIota activates src tyrosine kinases in vascular smooth muscle cells. The src family of tyrosine kinases are widely distributed non-receptor kinases that influence several signal transduction pathways. In the present study we evaluated the effect of a selective src family inhibitor, PP2, on aldosterone production using a human adrenocortical carcinoma-derived (H295R) cell line. Treatments for 6 or 48 h with PP2 (0.3 microM-10 microM) inhibited basal, Ang IotaIota, K(+) and dibutyryladenosine cyclic monophosphate (dbcAMP) stimulation of aldosterone production in a concentration-dependent manner. PP2 did not affect cell viability at any of the concentrations tested. Moreover, time course studies using PP2 (10 microM) for 6, 12, 24, and 48 h revealed a time-dependent inhibition of aldosterone production. Inhibition by PP2 (0.3-10 microM) was also observed for the metabolism of 22R-hydroxycholesterol (22R-OHChol) to aldosterone in H295R cells. Since 22R-OHChol is a substrate for cytochrome P450 side-chain cleavage enzyme (CYP11A) that does not require steroidogenic acute regulatory (StAR) protein for transport to the inner mitochondrial membrane, these results suggest that PP2 inhibition occurred beyond the rate-limiting step in aldosterone synthesis. Genistein, a non-specific tyrosine kinase inhibitor also blocked aldosterone production, but the inhibition was the result of a non-specific effect on 3beta-hydroxysteroid dehydrogenase (3betaHSD). In contrast, PP2 did not appear to act as a direct inhibitor of 3betaHSD activity. To further investigate the site of PP2 action, we examined its effect on H295R cell metabolism of [(14)C]progesterone using thin layer chromatography. PP2 treatment for 48 h caused an increase in the conversion of progesterone to 17alpha-hydroxyprogesterone. To determine if this apparent increase in 17alpha-hydroxylase activity was due to increased transcript, we examined the effect of PP2 on CYP17 mRNA. PP2 treatment caused an increase in CYP17 mRNA without an effect on 3betaHSD mRNA levels. Inhibition of protein synthesis with cycloheximide increased basal levels of CYP17 mRNA levels and blocked the induction observed by PP2. This suggests that new protein synthesis is a necessary part of PP2 induction of CYP17. Taken together these data suggest that the src tyrosine kinase inhibitor, PP2, is a potent inhibitor of aldosterone production. One mechanism for the inhibition is through an induction of CYP17 mRNA and enzyme activity. Src tyrosine kinases, therefore, may be involved with the promotion of a glomerulosa phenotype through the inhibition of CYP17 expression.