Arterial stiffening occurs with age and is associated with lack of exercise. Notably both age and lack of exercise are major cardiovascular risk factors. While it is well established that bulk arterial stiffness increases with age, more recent data suggest that the intima, the innermost arterial layer, also stiffens during aging. Micro-scale mechanical characterization of individual layers is important because cells primarily sense the matrix that they are in contact with and not necessarily the bulk stiffness of the vessel wall. To investigate the relationship between age, exercise, and subendothelial matrix stiffening, atomic force microscopy was utilized here to indent the subendothelial matrix of the thoracic aorta from young, aged-sedentary, and aged-exercised mice, and elastic modulus values were compared to conventional pulse wave velocity measurements. The subendothelial matrix elastic modulus was elevated in aged-sedentary mice compared to young or aged-exercised mice, and the macro-scale stiffness of the artery was found to linearly correlate with the subendothelial matrix elastic modulus. Notably, we also found that with age, there exists an increase in the point-to-point variations in modulus across the subendothelial matrix, indicating non-uniform stiffening. Importantly, this heterogeneity is reversible with exercise. Given that vessel stiffening is known to cause aberrant endothelial cell behavior, and the spatial heterogeneities we find exist on a length scale much smaller than the size of a cell, these data suggest that further investigation in the heterogeneity of the subendothelial matrix elastic modulus is necessary to fully understand the effects of physiological matrix stiffening on cell function.
Urinary RNA can be readily isolated and amplified for prostate cancer biomarker analysis. Individual patients had unique set of transcripts derived from their tumor.
The elusive nature of assessing immunological processes in situ in organ transplantation is one of the major impediments to improve diagnostics and treatment. Here, we present a proof-of-concept study using multiplexed in situ hybridization (ISH) (RNAscope) to detect low-abundance cytokines in formalin-fixed paraffin-embedded (FFPE) human transplant kidney biopsies in combination with immunofluorescence (IF) for cell phenotyping. We show that a multiplex IF and ISH (mIFISH) assay is feasible to identify the cellular source of cytokines and chemokines (tumor necrosis factor-α, interferon-γ, and CXCL9) in FFPE transplant kidney biopsies and that quantification of the mRNA and protein signal is also possible at single-cell resolution in the context of tissue complexity. Furthermore, the mIFISH assay allows precise quantitative assessment of tubulitis, one of the key morphological correlates of alloimmune injury. Simultaneous in situ identification and quantification of multiple cellular phenotypes and mRNA expression of proinflammatory cytokines in FFPE tissues offer a novel insight into the biology of alloimmune injury in kidney transplantation and may contribute to improved diagnostic accuracy and patient care.
The introduction of calcineurin inhibitors (CNIs) ushered a new era of renal transplantation by dramatically improving short-term allograft survival. 1,2 However, CNIs also became an additional contributor to late allograft loss due to their deleterious toxicity profile. 3 Since the late 1990s, there have been ongoing efforts to minimize CNI use or replace them with less toxic agents such as mycophenolate mofetil (MMF) with or without mechanistic target of rapamycin (mTOR) inhibitors and steroid. 4,5 The most recent and, so far, most promising attempt was the introduction of belatacept 6 (a second-generation CTLA4-Ig). Belatacept acts by blocking the
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