Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue-Resident Macrophages In Vivo in Mice.

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

Modest changes in Spi1 dosage reveal the potential for altered microglial function as seen in Alzheimer's disease.

Genetic association studies have identified multiple variants at the SPI1 locus that modify risk and age of onset for Alzheimer's Disease (AD). Reports linking risk variants to gene expression suggest that variants denoting higher SPI1 expression are likely to have an earlier AD onset, and several other AD risk genes contain PU.1 binding sites in the promoter region. Overall, this suggests the level of SPI1 may alter microglial phenotype potentially impacting AD. This study determined how the microglial transcriptome was altered following modest changes to Spi1 expression in primary mouse microglia. RNA-sequencing was performed on microglia with reduced or increased Spi1/PU.1 expression to provide an unbiased approach to determine transcriptomic changes affected by Spi1. In summary, a reduction in microglial Spi1 resulted in the dysregulation of transcripts encoding proteins involved in DNA replication pathways while an increased Spi1 results in an upregulation of genes associated with immune response pathways. Additionally, a subset of 194 Spi1 dose-sensitive genes was identified and pathway analysis suggests that several innate immune and interferon response pathways are impacted by the concentration of Spi1. Together these results suggest Spi1 levels can alter the microglial transcriptome and suggests interferon pathways may be altered in individuals with AD related Spi1 risk SNPs.

Craniofacial and Long Bone Development in the Context of Distraction Osteogenesis.

BACKGROUND: Bone retains regenerative potential into adulthood, and surgeons harness this plasticity during distraction osteogenesis. The underlying biology governing bone development, repair, and regeneration is divergent between the craniofacial and appendicular skeleton. Each type of bone formation is characterized by unique molecular signaling and cellular behavior. Recent discoveries have elucidated the cellular and genetic processes underlying skeletal development and regeneration, providing an opportunity to couple biological and clinical knowledge to improve patient care. METHODS: A comprehensive literature review of basic and clinical literature regarding craniofacial and long bone development, regeneration, and distraction osteogenesis was performed. RESULTS: The current understanding in craniofacial and long bone development and regeneration is discussed, and clinical considerations for the respective distraction osteogenesis procedures are presented. CONCLUSIONS: Distraction osteogenesis is a powerful tool to regenerate bone and thus address a number of craniofacial and appendicular skeletal deficiencies. The molecular mechanisms underlying bone regeneration, however, remain elusive. Recent work has determined that embryologic morphogen gradients constitute important signals during regeneration. In addition, striking discoveries have illuminated the cellular processes underlying mandibular regeneration during distraction osteogenesis, showing that skeletal stem cells reactivate embryologic neural crest transcriptomic processes to carry out bone formation during regeneration. Furthermore, innovative adjuvant therapies to complement distraction osteogenesis use biological processes active in embryogenesis and regeneration. Additional research is needed to further characterize the underlying cellular mechanisms responsible for improved bone formation through adjuvant therapies and the role skeletal stem cells play during regeneration.

Doxycycline Reduces Scar Thickness and Improves Collagen Architecture.

OBJECTIVE: To investigate the effects of local doxycycline administration on skin scarring. BACKGROUND: Skin scarring represents a major source of morbidity for surgical patients. Doxycycline, a tetracycline antibiotic with off-target effects on the extracellular matrix, has demonstrated antifibrotic effects in multiple organs. However, doxycycline's potential effects on skin scarring have not been explored in vivo. METHODS: Female C57BL/6J mice underwent dorsal wounding following an established splinted excisional skin wounding model. Doxycycline was administered by local injection into the wound base following injury. Wounds were harvested upon complete wound closure (postoperative day 15) for histological examination and biomechanical testing of scar tissue. RESULTS: A one-time dose of 3.90 mM doxycycline (2 mg/mL) within 12 hours of injury was found to significantly reduce scar thickness by 24.8% (P < 0.0001) without compromising tensile strength. The same effect could not be achieved by oral dosing. In doxycycline-treated scar matrices, collagen I content was significantly reduced (P = 0.0317) and fibers were favorably arranged with significantly increased fiber randomness (P = 0.0115). Common culprits of altered wound healing mechanics, including angiogenesis and inflammation, were not impacted by doxycycline treatment. However, engrailed1 profibrotic fibroblasts, responsible for scar extracellular matrix deposition, were significantly reduced with doxycycline treatment (P = 0.0005). CONCLUSIONS: Due to the substantial improvement in skin scarring and well-established clinical safety profile, locally administered doxycycline represents a promising vulnerary agent. As such, we favor rapid translation to human patients as an antiscarring therapy.

Angiotensin-converting enzyme in cerebrospinal fluid and risk of brain atrophy.

BACKGROUND: Higher angiotensin-converting enzyme (ACE) activity might increase the risk of Alzheimer's disease by increasing blood pressure, and subsequent development of cerebral small vessel disease (CSVD). Yet, it may also decrease this risk, as it functions to degrade amyloid-ß, thereby reducing brain atrophy. OBJECTIVE: To examine the cross-sectional associations of serum and cerebrospinal fluid (CSF) ACE protein levels and activity with brain atrophy and CSVD in a memory clinic cohort. METHODS: In 118 subjects from the memory clinic based Amsterdam Dementia Cohort (mean age 66 ± 8 years), ACE protein levels (ng/ml) and activity in CSF and serum were investigated. Poisson regression analyses were used to associate ACE measurements with rated global cortical atrophy, medial temporal lobe atrophy, lacunar infarcts, white matter hyperintensities, and microbleeds on brain MRI. RESULTS: Higher CSF ACE activity was associated with a reduced risk of global brain atrophy. The relative risk (95% CI) of having global cortical atrophy ≥2 per SD increase in CSF ACE activity was 0.67 (0.49; 0.93). ACE levels were not significantly related to measures of CSVD. CONCLUSIONS: These results show that high ACE might have protective effects on the brain. This could suggest that ACE inhibitors, which may lower CSF ACE levels, are not preferred as antihypertensive treatment in patients at risk for Alzheimer's disease.

The association of angiotensin-converting enzyme with biomarkers for Alzheimer's disease.

INTRODUCTION: Lower angiotensin-converting enzyme (ACE) activity could increase the risk of Alzheimer's disease (AD) as ACE functions to degrade amyloid-ß (Aß). Therefore, we investigated whether ACE protein and activity levels in cerebrospinal fluid (CSF) and serum were associated with CSF Aß, total tau (tau) and tau phosphorylated at threonine 181 (ptau). METHODS: We included 118 subjects from our memory clinic-based Amsterdam Dementia Cohort (mean age 66 ± 8 years) with subjective memory complaints (n = 40) or AD (n = 78), who did not use antihypertensive drugs. We measured ACE protein levels (ng/ml) and activity (RFU) in CSF and serum, and amyloid ß1-42, tau and ptau (pg/ml) in CSF. RESULTS: Cross-sectional regression analyses showed that ACE protein level and activity in CSF and serum were lower in patients with AD compared to controls. Lower CSF ACE protein level, and to a lesser extent serum ACE protein level and CSF ACE activity, were associated with lower CSF Aß, indicating more brain Aß pathology; adjusted regression coefficients (B) (95% CI) per SD increase were 0.09 (0.04; 0.15), 0.06 (0.00; 0.12) and 0.05 (0.00; 0.11), respectively. Further, lower CSF ACE protein level was associated with lower CSF tau and ptau levels; adjusted B's (95% CI) per SD increase were 0.15 (0.06; 0.25) and 0.17 (0.10; 0.25), respectively. CONCLUSIONS: These results strengthen the hypothesis that ACE degrades Aß. This could suggest that lowering ACE levels by for example ACE-inhibitors might have adverse consequences for patients with, or at risk for AD.