E. Alfonso Romero-Sandoval, MD, PhD, Wake Forest University (formerly Presbyterian College)
PROJECT TITLE: Finding CD163 dependent pathways in diabetic macrophages using bioinformatics
Our project has the potential to develop new drugs or therapeutic strategies to improve wound healing in patients suffering diabetes. Peripheral neuropathy is a common complication of diabetes, and foot ulcers derive from diabetic neuropathy. These ulcers are the major cause of foot amputation. Therefore, our studies have the potential to have a significant impact in the quality of life of patients with diabetes.
The aims of this proposal were:
- Perform a whole-transcriptome analysis (total RNA sequencing) and differential gene/pathway expression determination workflow using bioinformatics in macrophages from diabetic patients with and without neuropathy.
- Identify target genes/pathways that are modulated by CD163 in diabetic macrophages.
- Utilize these data to prepare a proposal for NIH funding (R15, R21 or R01).
We recruited three diabetic patients without neuropathy, three diabetic patients with neuropathy, and three healthy volunteers. Monocytes were successfully collected and differentiated into macrophages in vitro, RNA was isolated, and quality and quantity were was measured. These samples were processed for a meta-genome sequencing and bioinformatics analyses at Clemson University (Dr. Christopher Saski). These data were compared with a meta-genome analysis performed previously (project from other funding sources) in primary macrophages from healthy individuals undergone CD163 gene induction and respective control group (Figure 1).
Figure 1. Table and Volcano plot of primary macrophages from healthy individuals versus primary macrophages from diabetic patients, and versus primary macrophages from diabetic neuropathy patients (left panel). Volcano plot of primary macrophages from healthy individuals with no stimulation (control) versus macrophages with LPS stimulation, and macrophages with LPS stimulation transfected with pEmpty versus pCD163 (right panel).
We have observed that macrophages overexpressing CD163 induces a more efficient in vitro wound healing by interacting with skin cells. This does not occur with diabetic macrophages, or with diabetic macrophages with CD163 overexpression. We therefore pursue genes that are related to cell migration that are related or unrelated to CD163. We identified cell migration genes in both set of data to determine the potential role of CD163 dependent or non-dependent genes in diabetic macrophages. Figure 2 shows the identified genes (red circled) that are altered in diabetic macrophages that are not dependent upon CD163 signaling pathway and may explain the deficiency of diabetic macrophages in inducing wound healing interactions with skin cells.
Figure 2. Differentially expressed genes related to cell migration represented as fold-change of healthy vs. diabetic (H vs. D), control vs. LPS stimulated (C vs. LPS), and LPS+pEmpty vs. LPS+pCD163 (LPS vs. CD163).
We conclude the following:
- Diabetic macrophages possess a dysfunctional wound healing function that does not depend on CD163.
- We preliminary identified target genes that potentially play a role in migration that are not dependent on CD163 in diabetic macrophages.
- We identified CD163-dependent genes that play a role in cell migration under inflammatory conditions that are not related to diabetes.
Our future directions are focused on an NIH grant that will propose to follow up the identified genes in diabetic macrophages using. A gene modulation (induction or silencing) with functional outcomes will be proposed as well. If collaborators are engaged, we could apply our interventions using in vivo models of diabetic wound healing.
In addition, we are currently conducting additional bioinformatics analysis to identify other functional pathways (inflammation, phagocytosis, apoptosis, mitochondrial metabolism, etc.) to potentially pursue specific genes for different functional outcomes. We are also preparing a manuscript pertaining to our current data, and an NIH grant (R21) to continue with the abovementioned studies.
SC INBRE Bioinformatics Pilot Project Program funding was instrumental in generating these preliminary data because this approach (gene sequencing and bioinformatics analysis) is expensive, but powerful. We had some previous data that trigger this idea. but we did not have the sufficient funding to pursue it. SC INBRE bioinformatics grant has made possible the completion of these studies and enabled the preparation of a subsequent NIH grant application.
April 6, 2018