Hu, Nan

Abstract Keratoconus (KCN) is a leading cause for cornea grafting worldwide. Keratoconus is a multifactorial disease that causes progressive thinning of the cornea and whose etiology is poorly understood. Several studies have used proteomics on patient tear fluids to identify potential biomarkers. However, proteome of the cornea itself has not been investigated fully. We report here new findings from a case–control study using multiplexed mass spectrometry (MS) on individual (unpooled) corneas to gain deeper insights into proteins and biomarkers relevant to keratoconus. We employed a high-pressure approach to extract total protein from individual corneas from five cases and five controls, followed by trypsin digestion and tandem mass tag (TMT) labeling. The MS-derived data were searched using the Human NCBI RefSeq protein database v92, with peptides and proteins filtered at 1% false discovery rate. A total of 3132 proteins were detected, of which 627 were altered significantly ( p ≤ 0.05) in keratoconus corneas. The increases were overwhelmingly in the mTOR/PI3/AKT signal-mediated regulations of cell survival and proliferation, nonsense-mediated decay of transcripts, and proteasomal pathways. The decreases were in several extracellular matrix proteins and in many members of the complement system. Importantly, this multiplexed proteomic study of keratoconus corneas identified, to our knowledge, the largest number of corneal proteins. The novel findings include changes in pathways that regulate transcript stability, proteasomal degradation, and the complement system in corneas with keratoconus. These observations offer new prospects toward future discovery of novel molecular targets for diagnostic and therapeutic innovations for patients with keratoconus.

Peripheral artery disease (PAD) in the lower extremities often leads to intermittent claudication. In the present study, we proposed a low-dose DCE MRI protocol for quantifying calf muscle perfusion stimulated with plantar flexion and multiple new metrics for interpreting perfusion maps, including the ratio of gastrocnemius over soleus perfusion (G/S; for assessing the vascular redistribution between the two muscles) and muscle perfusion normalized by whole body perfusion (for quantifying the muscle’s active hyperemia). Twenty-eight human subjects participated in this Institutional Review Board-approved study, with 10 healthy subjects ( group A) for assessing interday reproducibility and 8 healthy subjects ( group B) for exploring the relationship between plantar-flexion load and induced muscle perfusion. In a pilot group of five elderly healthy subjects and five patients with PAD ( group C), we proposed a protocol that measured perfusion for a low-intensity exercise and for an exhaustion exercise in a single MRI session. In group A, perfusion estimates for calf muscles were highly reproducible, with correlation coefficients of 0.90–0.93. In group B, gastrocnemius perfusion increased linearly with the exercise workload ( P < 0.05). With the low-intensity exercise, patients with PAD in group C showed substantially lower gastrocnemius perfusion compared with elderly healthy subjects [43.4 (SD 23.5) vs. 106.7 (SD 73.2) ml·min−1·100 g−1]. With exhaustion exercise, G/S [1.0 (SD 0.4)] for patients with PAD was lower than both its low-intensity level [1.9 (SD 1.3)] and the level in elderly healthy subjects [2.7 (SD 2.1)]. In conclusion, the proposed MRI protocol and the new metrics are feasible for quantifying exercise-induced muscle hyperemia, a promising functional test of PAD.NEW & NOTEWORTHY To quantitatively map exercise-induced hyperemia in calf muscles, we proposed a high-resolution MRI method shown to be highly reproducible and sensitive to exercise load. With the use of low contrast, it is feasible to measure calf muscle hyperemia for both low-intensity and exhaustion exercises in a single MRI session. The newly proposed metrics for interpreting perfusion maps are promising for quantifying intermuscle vascular redistribution or a muscle’s active hyperemia.