Sarah Melissa Cutler

Engineering cell adhesive surfaces that direct integrin α5β1 binding using a recombinant fragment of fibronectin

Integrin receptors mediate cell adhesion to extracellular matrices and trigger signals that direct cell function. While many integrins bind to the arginine-glycine-aspartic acid (RGD) motif present in numerous extracellular proteins, integrin alpha(5)beta(1) requires both the PHSRN synergy site in the 9th and the RGD site in the 10th type III repeat of fibronectin (FN). Binding of alpha(5)beta(1) to FN is critical to many cellular processes, including osteoblast and myoblast differentiation. This work focused on engineering integrin-specific bioadhesive surfaces by immobilizing a recombinant FN fragment (FNIII(7-10)) encompassing the alpha(5)beta(1) binding domains of FN. Model hybrid surfaces were engineered by immobilizing FNIII(7-10) onto passively adsorbed, non-adhesive albumin. Homo- and hetero-bifunctional crosslinkers of varying spacer-arm length targeting either the cysteine or lysine groups on FNIII(7-10) were investigated in ELISA and cell adhesion assays to optimize immobilization densities and activity. FN-mimetic surfaces presenting controlled densities of FNIII(7-10) were generated by varying the concentration of FNIII(7-10) in the coupling solution at a constant crosslinker concentration. Cells adhered to these functionalized surfaces via integrin alpha(5)beta(1) and blocking with integrin-specific antibodies completely eliminated adhesion. In addition, adherent cells spread and assembled focal adhesions containing alpha(5)beta(1), vinculin, and talin. This biomolecular engineering strategy represents a robust approach to increase biofunctional activity and integrin specificity of biomimetic materials.

Neurosteroids reduce inflammation after TBI through CD55 induction

The inflammatory cascade that follows traumatic brain injury may lead to secondary cell death and can impede recovery of function. Complement factors and their convertases are increased in glia after brain injury and lead to the production of inflammatory products that kill vulnerable neurons. Progesterone and its metabolite allopregnanolone (5alpha-pregnan-3beta-ol-20-one) have been shown to reduce the expression of inflammatory cytokines in the acute stages of brain injury, although how they do this is not completely understood. In this study we show that both progesterone and allopregnanolone treatments enhance the production of CD55 following contusion injuries of the cerebral cortex in rats. CD55, a single-chain type 1 cell surface protein, is a potent inhibitor of the complement convertases which are activators of the inflammatory cascade. The increased expression of CD55 could be an important mechanism by which steroids help to reduce the cerebral damage caused by inflammation.

Tapered progesterone withdrawal enhances behavioral and molecular recovery after traumatic brain injury

Systemic injections of the neurosteroid progesterone improve cognitive recovery after traumatic brain injury (TBI) and stroke, and decrease molecular indicators of neuronal damage. Suddenly withdrawing progesterone after repeated dosing (PW) exacerbates ischemia and causes increased anxiety, seizure susceptibility, and excitotoxicity. Adult male Sprague-Dawley rats received either bilateral medial frontal cortex contusions or sham surgery. Injections were administered at 1 and 6 h post-injury, then every 24 h for 7 days. Vehicle-treated rats received 2-hydroxypropyl-beta-cyclodextrin (HBC). Acute PW (AW) rats received a full 16 mg/ml of progesterone for 7 days, and tapered PW (TW) rats received 5 days at full dosage, then 2 days with progressively halved dosages. Anxiety behaviors were observed pre- and post-surgery, and compared to levels at the peak of withdrawal. AW rats with lesions exhibited significantly more anxiety than any other treatment group, while both lesion- and sham-operated TW rats were indistinguishable from vehicle-treated intact animals. After behavioral tests were complete, the brains were extracted and prepared for Western blotting. TNFalpha, cFos, Caspase-3, and NFkappaB, among others, were investigated. While all progesterone treatments resulted in improved molecular recovery, TW animals had significantly fewer active markers for apoptosis and inflammation than AW animals. In conclusion, although progesterone treatment decreases inflammation and apoptosis, acute withdrawal increases activity in some apoptotic and inflammatory pathways and increases anxiety behavior during the acute healing phase. A tapered withdrawal of the hormone further enhances short-term recovery after TBI.

The enantiomer of progesterone acts as a molecular neuroprotectant after traumatic brain injury

Previous work shows that neurosteroid enantiomers activate specific molecular receptors that relay neuroprotection. However, the actions of the enantiomer of progesterone (ent-PROG) at the PROG receptor (PR) are unknown. PR binding and transcriptional assays were performed to determine the actions of ent-PROG at the classical PR. Additionally, the neuroprotective effects of ent-PROG in traumatic brain injury (TBI) were investigated and compared to the actions of PROG and its metabolite allopregnanolone (ALLO), both of which have been shown to have neuroprotective properties when given after TBI. Binding studies performed in COS cells over-expressing the PR showed that ent-PROG inhibited PROG binding to the PR. In contrast, ent-PROG did not activate PR-mediated transcription. Rats received bilateral medial frontal cortex injury followed by treatments at 1, 6, 24 and 48h with PROG, ALLO or ent-PROG. Brains were processed for edema, protein and enzyme activity. ent-PROG treatment in vivo decreased cerebral edema, cell death mediators, inflammatory cytokines, and reactive gliosis, and increased antioxidant activity. These findings suggest that the progestin-mediated pro-survival response seen with TBI is regulated either independently of the classical PR or via nongenomic PR-regulated actions.

Slow-release and injected progesterone treatments enhance acute recovery after traumatic brain injury

The benefits of continuous progesterone release via subcutaneous silastic capsule implants were compared to daily subcutaneous injections in a rat model of traumatic brain injury (TBI). Adult male Sprague-Dawley rats received either bilateral frontal cortex contusions or sham surgery. Rats were injected with progesterone or vehicle at 1 and 6 h post-injury, then once every 24 h for six days with tapering of the dose over the final two treatments. Progesterone-packed silastic capsules were implanted post-injury while the animals were anesthetized. Behavioral assays for anxiety and locomotor activity were evaluated pre- and post-TBI. Brains were extracted eight days post-TBI and prepared for molecular assays. Decreased GABAA-4 levels complemented a decrease in anxiety behaviors on the Elevated Plus Maze for capsule compared to progesterone-injected animals prior to daily injections. All groups with implanted capsules increased locomotor activity compared to those given progesterone injections. In conclusion, steady-state progesterone treatment after TBI decreases edema and anxiety and increases activity, thus enhancing behavioral recovery. A continuous mode of pharmacological administration may prove to be more beneficial in translational and clinical testing than bolus injections over the same period of time.

Tapered progesterone withdrawal promotes long-term recovery following brain trauma

We previously demonstrated that after traumatic brain injury (TBI), acute progesterone withdrawal (AW) causes an increase in anxiety behaviors and cerebro-cellular inflammation compared to tapered progesterone withdrawal (TW). Our current study investigates the behavioral and cellular effects of AW two weeks after termination of treatments to determine the longer-term influence of withdrawal after injury. Adult, male Sprague-Dawley rats received either bilateral frontal cortex contusion (L) or sham (S) surgery. Rats were injected at 1 and 6 h post-injury, then every 24 h for six days. Vehicle (V)-treated rats were given 9 injections of 22.5% cyclodextrin, whereas AW rats received 9 injections of 16 mg/kg progesterone and TW rats received 7 injections of P at 16 mg/kg, followed by one at 8 mg/kg and one at 4 mg/kg. On day 8, sensory neglect and locomotor activity tests were initiated. Animals were killed 22 days post-TBI and the brains prepared for either molecular or histological analysis. Western blotting revealed increased brain-derived neurotrophic factor (BDNF) and heat shock protein 70 (HSP70) in TW vs. AW animals. P53 was increased in VL animals, whereas all progesterone-treated groups were equivalent to shams. TW animals had markedly decreased sensory neglect compared to AW animals and increased center time in locomotor activity assays. In addition, lesion reconstruction revealed a decreased lesion size for TWL over AWL over VL animals. Glial fibrillary acidic protein (GFAP) immunofluorescent staining followed this pattern as well. In conclusion, after TBI, AW affects select behaviors and molecular markers in the chronic recovery period.

Vitamin D deficiency reduces the benefits of progesterone treatment after brain injury in aged rats

Administration of the neurosteroid progesterone (PROG) has been shown to be beneficial in a number of brain injury models and in two recent clinical trials. Given widespread vitamin D deficiency and increasing traumatic brain injuries (TBIs) in the elderly, we investigated the interaction of vitamin D deficiency and PROG with cortical contusion injury in aged rats. Vitamin D deficient (VitD-deficient) animals showed elevated inflammatory proteins (TNFα, IL-1β, IL-6, NFκB p65) in the brain even without injury. VitD-deficient rats with TBI, whether given PROG or vehicle, showed increased inflammation and greater open-field behavioral deficits compared to VitD-normal animals. Although PROG was beneficial in injured VitD-normal animals, in VitD-deficient subjects neurosteroid treatment conferred no improvement over vehicle. A supplemental dose of 1,25-dihydroxyvitamin D(3) (VDH) given with the first PROG treatment dramatically improved results in VitD-deficient rats, but treatment with VDH alone did not. Our results suggest that VitD-deficiency can increase baseline brain inflammation, exacerbate the effects of TBI, and attenuate the benefits of PROG treatment; these effects may be reversed if the deficiency is corrected.

Progesterone and its metabolite allopregnanolone differentially regulate hemostatic proteins after traumatic brain injury

Our laboratory has shown in numerous experiments that the neurosteroids progesterone (PROG) and allopregnanolone (ALLO) improve molecular and functional outcomes after traumatic brain injury (TBI). As coagulopathy is an important contributor to the secondary destruction of nervous tissue, we hypothesized that PROG and ALLO administration may also have a beneficial effect on coagulation protein expression after TBI. Adult male Sprague—Dawley rats were given bilateral contusions of the medial frontal cortex followed by treatments with PROG (16 mg/kg), ALLO (8 mg/kg), or vehicle (22.5% hydroxypropyl-β-cyclodextrin). Controls received no injury or injections. Progesterone generally maintained procoagulant (thrombin, fibrinogen, and coagulation factor XIII), whereas ALLO increased anticoagulant protein expression (tissue-type plasminogen activator, tPA). In addition, PROG significantly increased the ratio of tPA bound to neuroserpin, a serine protease inhibitor that can reduce the activity of tPA. Our findings suggest that in a model of TBI, where blood loss may exacerbate injury, it may be preferable to treat patients with PROG, whereas it might be more appropriate to use ALLO as a treatment for thrombotic stroke, where a reduction in coagulation would be more beneficial.