Single cell transcriptomic landscape of diabetic foot ulcers.

Diabetic foot ulceration (DFU) is a devastating complication of diabetes whose pathogenesis remains incompletely understood. Here, we profile 174,962 single cells from the foot, forearm, and peripheral blood mononuclear cells using single-cell RNA sequencing. Our analysis shows enrichment of a unique population of fibroblasts overexpressing MMP1, MMP3, MMP11, HIF1A, CHI3L1, and TNFAIP6 and increased M1 macrophage polarization in the DFU patients with healing wounds. Further, analysis of spatially separated samples from the same patient and spatial transcriptomics reveal preferential localization of these healing associated fibroblasts toward the wound bed as compared to the wound edge or unwounded skin. Spatial transcriptomics also validates our findings of higher abundance of M1 macrophages in healers and M2 macrophages in non-healers. Our analysis provides deep insights into the wound healing microenvironment, identifying cell types that could be critical in promoting DFU healing, and may inform novel therapeutic approaches for DFU treatment.

Integrated Skin Transcriptomics and Serum Multiplex Assays Reveal Novel Mechanisms of Wound Healing in Diabetic Foot Ulcers.

Nonhealing diabetic foot ulcers (DFUs) are characterized by low-grade chronic inflammation, both locally and systemically. We prospectively followed a group of patients who either healed or developed nonhealing chronic DFUs. Serum and forearm skin analysis, both at the protein expression and the transcriptomic level, indicated that increased expression of factors such as interferon-γ (IFN-γ), vascular endothelial growth factor, and soluble vascular cell adhesion molecule-1 were associated with DFU healing. Furthermore, foot skin single-cell RNA sequencing analysis showed multiple fibroblast cell clusters and increased inflammation in the dorsal skin of patients with diabetes mellitus (DM) and DFU specimens compared with control subjects. In addition, in myeloid cell DM and DFU upstream regulator analysis, we observed inhibition of interleukin-13 and IFN-γ and dysregulation of biological processes that included cell movement of monocytes, migration of dendritic cells, and chemotaxis of antigen-presenting cells pointing to an impaired migratory profile of immune cells in DM skin. The SLCO2A1 and CYP1A1 genes, which were upregulated at the forearm of nonhealers, were mainly expressed by the vascular endothelial cell cluster almost exclusively in DFU, indicating a potential important role in wound healing. These results from integrated protein and transcriptome analyses identified individual genes and pathways that can potentially be targeted for enhancing DFU healing.

Topical Treatment of Skin Injury Inflicted in Mice by X-Ray Irradiation.

BACKGROUND/AIMS: There is no treatment, without side effects, efficiently preventing or curing skin burns, caused by radiotherapy. A new experimental topical treatment protocol was assessed in mice receiving orthovoltage X-rays at an equivalent dose to that applied to human breast cancer patients in conventional radiotherapy. METHODS: SKH-HR2 female hairless mice were irradiated on their dorsum with a total dose of 4,300 cGy during a 1-month period (20 fractions). The treatment group received a combination of 3 topical products, an oil-in-water cream, a gel containing Pinus halepensis bark aqueous extract, and an ointment containing olive oil extract of the marine isopod Ceratothoa oestroides. The positive control group was treated with a conventionally used commercial gel, whereas the negative control group did not receive any topical treatment. Skin alterations were evaluated by macroscopic examinations, measurements of transepidermal water loss (TEWL), melanin content, erythema intensity, hydration, and histopathology assessment. RESULTS: Sixty days after radiation, TEWL and hydration values were abnormal and elements of acute, chronic, and granulomatous inflammation were present in all cases. The severest damage was detected in the deeper dermis. Treatment showed a comparatively beneficial effect on chronic and granulomatous inflammation while positive control was beneficial on acute inflammation. CONCLUSION: Skin anti-inflammatory treatment was the most effective but must be applied for several months. Further preclinical studies should be conducted, assimilating a human cancer radiation therapeutic schema with the aim of optimizing skin inflammation treatment.

Substance P promotes wound healing in diabetes by modulating inflammation and macrophage phenotype.

Diabetic foot ulceration is a major complication of diabetes. Substance P (SP) is involved in wound healing, but its effect in diabetic skin wounds is unclear. We examined the effect of exogenous SP delivery on diabetic mouse and rabbit wounds. We also studied the impact of deficiency in SP or its receptor, neurokinin-1 receptor, on wound healing in mouse models. SP treatment improved wound healing in mice and rabbits, whereas the absence of SP or its receptor impaired wound progression in mice. Moreover, SP bioavailability in diabetic skin was reduced as SP gene expression was decreased, whereas the gene expression and protein levels of the enzyme that degrades SP, neutral endopeptidase, were increased. Diabetes and SP deficiency were associated with absence of an acute inflammatory response important for wound healing progression and instead revealed a persistent inflammation throughout the healing process. SP treatment induced an acute inflammatory response, which enabled the progression to the proliferative phase and modulated macrophage activation toward the M2 phenotype that promotes wound healing. In conclusion, SP treatment reverses the chronic proinflammatory state in diabetic skin and promotes healing of diabetic wounds.

Aliskiren improves vascular smooth muscle function in the skin microcirculation of type 2 diabetic patients with normal renal function.

OBJECTIVE: The objective of this paper is to study the effect of aliskiren on metabolic parameters and micro- and macrovascular reactivity in individuals diagnosed with or at high risk for developing type 2 diabetes mellitus (T2DM). RESEARCH DESIGN AND METHODS: We studied 47 T2DM and 41 at-risk individuals in a randomized, double-blinded, placebo-controlled trial. All subjects were treated with 150 mg aliskiren or placebo daily for 12 weeks. Twenty-six (55%) of T2DM and four (8%) at-risk subjects were also treated with angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers. RESULTS: Aliskiren treatment was associated with improvement in systolic and diastolic blood pressure and endothelium-independent vasodilation at the skin microcirculation in those with T2DM but not in those at risk. There were no incidences of hypotension and no significant changes in serum potassium or creatinine levels with aliskiren treatment in either study group. CONCLUSIONS: Aliskiren improves blood pressure and vascular smooth muscle function in the skin microcirculation of T2DM patients.

Increased skin inflammation and blood vessel density in human and experimental diabetes.

Systemic inflammation is associated with impaired wound healing in diabetes mellitus (DM) patients. Using immunohistochemistry techniques, the authors investigated changes in skin inflammation and skin blood vessels in human and experimental diabetes. Comparing to the non-DM human subjects, the total number of inflammatory cells per biopsy and the number of inflammatory cells around blood vessels, a strong indication of inflammation, were higher in DM subjects irrespective of their risk for developing diabetic foot ulcer. Inflammatory cell infiltration was robustly increased in all DM animal models compared with their non-DM controls. The number and density of blood vessels and CD31 positive proliferating endothelial cells around preexisting skin vessels was also higher in the DM patients. However, there were no differences in the skin blood flow between the non-DM and DM subjects. The number of skin blood vessels was also increased in the DM animals; however, these differences were less obvious than the ones observed for inflammatory cells. We conclude that skin inflammation and skin blood vessel density is increased in diabetic human subjects and in rodent and rabbit models of diabetes.

Evidence of sympathetic innervation and α1-adrenergic receptors of the long head of the biceps brachii tendon.

BACKGROUND: The primary purpose of this study was to investigate the sympathetic innervation of the long head of the biceps brachii tendon LHB via immunohistochemical staining for protein S-100 and neuropeptide Y (NPY) in patients with complex proximal humerus fractures, in individuals with chronic biceps tendinosis in the setting of large rotator cuff tears (RC), and in cadaveric samples with no previously reported shoulder pathology. METHODS: We investigated the presence of sympathetic innervation and α1-adrenergic receptors of the long head of the biceps brachii tendon (LHB) in patients with complex proximal humerus fractures and individuals with chronic biceps tendinosis in the setting of large rotator cuff tears (RC). The correlation of morphological features with immunohistochemical evidence of neural element presence was also investigated. Forty-one LHB tendon specimens were examined. Seventeen were harvested from patients who underwent hemiarthroplasty for proximal humerus fractures, 14 were from individuals with biceps tendinosis in the context of a large RC tear, and ten were from cadaveric controls with no previous shoulder pathology. Histologic examination was performed using hematoxylin and eosin. Immunohistochemistry was used to detect the expression of the protein S-100, neuropeptide Y, and α1-adrenergic receptors, as well as to characterize the potential neural differentiation of tendon cells. RESULTS: A strong correlation between the expression of NPY/S-100, α1-adrenergic/S-100, and α1-adrenergic/NPY was found. The LHB tendon has sympathetic innervation and α1-adrenergic receptors in acute and chronic pathological conditions. CONCLUSION: Our results provide useful guidance on the management of tendinosis and the handling of the LHB in hemiarthroplasties for fractures.

Mechanisms involved in the development and healing of diabetic foot ulceration.

We examined the role of vascular function and inflammation in the development and failure to heal diabetic foot ulcers (DFUs). We followed 104 diabetic patients for a period of 18.4 ± 10.8 months. At the beginning of the study, we evaluated vascular reactivity and serum inflammatory cytokines and growth factors. DFUs developed in 30 (29%) patients. DFU patients had more severe neuropathy, higher white blood cell count, and lower endothelium-dependent and -independent vasodilation in the macrocirculation. Complete ulcer healing was achieved in 16 (53%) patients, whereas 13 (47%) patients did not heal. There were no differences in the above parameters between the two groups, but patients whose ulcers failed to heal had higher tumor necrosis factor-α, monocyte chemoattractant protein-1, matrix metallopeptidase 9 (MMP-9), and fibroblast growth factor 2 serum levels when compared with those who healed. Skin biopsy analysis showed that compared with control subjects, diabetic patients had increased immune cell infiltration, expression of MMP-9, and protein tyrosine phosphatase-1B (PTP1B), which negatively regulates the signaling of insulin, leptin, and growth factors. We conclude that increased inflammation, expression of MMP-9, PTP1B, and aberrant growth factor levels are the main factors associated with failure to heal DFUs. Targeting these factors may prove helpful in the management of DFUs.

Correlation between histologic assessment and fluorescence in situ hybridization using MelanoSITE in evaluation of histologically ambiguous melanocytic lesions.

CONTEXT: The 4-probe, multicolor, fluorescence in situ hybridization (FISH) panel targeting chromosomes 6 and 11 has shown promising sensitivity and specificity in distinguishing between benign nevi and malignant melanoma. Only a few studies have assessed the potential utility of FISH in classification of histologically ambiguous melanocytic lesions. In the United States, this assay is exclusively licensed to NeoGenomics Laboratories (Irvine, California), which provides the technical component and has developed an innovative service (MelanoSITE) allowing pathologists to interpret FISH results using a dedicated Web portal. Thus far, use of MelanoSITE as a diagnostic adjunct in the diagnosis of melanocytic lesions has not, to our knowledge, been reported in the literature. OBJECTIVE: To analyze 1.5 years of experience with the MelanoSITE melanoma FISH assay in the evaluation of histologically ambiguous lesions in the context of second opinion and routine dermatopathology practice. DESIGN: A prospective histologic/FISH correlation study of 140 cases. RESULTS: Twenty-seven percent of abnormal FISH results were false-positive results because of tetraploidy. After correcting for known false-positive results, all lesions considered atypical nevi showed normal FISH signals. Abnormal FISH signals were reported in 30% of lesions considered histologically borderline and in 48% of lesions in which a diagnosis of melanoma was favored. CONCLUSIONS: Four-probe, multicolor FISH results for melanoma correlate with the microscopic assessments of histologically ambiguous lesions. Pathologists using MelanoSITE must be aware of the high rate of false-positive results from tetraploidy.

Syringotropic melanoma: a variant of melanoma with prominent involvement of eccrine apparatus and risk of deep dermal invasion.

We report 7 cases which can be regarded as a syringotropic melanoma-a unique presentation of melanoma defined as melanoma spreading within the eccrine apparatus into the reticular dermis and/or subcutaneous tissue deeper than any (if present) associated invasive melanoma. Six patients were females, and 1 was a male. Their ages ranged from 32 to 85 years old (average 63). The lesions showed a wide site distribution, occurring on the extremities (4), trunk (2), and head and neck (1). Five melanomas were superficial spreading type; 1 was acral lentiginous type; 1 was unclassified. Four lesions (57%) invaded from within eccrine apparatus at a depth and anatomical level greater than that of an adjacent conventional invasive melanoma arising from the surface epidermis. In 1 lesion, which presented clinically as a pigmented macule, deep dermal syringocentric invasive tumor was the only site of invasion and tumorigenic growth. Thus, this variant of melanoma carries a significant risk of syringocentric deep dermal invasion, which may be unsuspected clinically but must be detected on histological examination to provide the most accurate prognosis and staging information.

Cutaneous alterations in diabetes mellitus.

 Dermatological problems occur with increased frequency in individuals with diabetes mellitus (DM). Cutaneous manifestations may be the first presenting sign of DM or even precede the diagnosis by many years. The main changes in the skin are due to alterations of microcirculation, the nervous system, and collagen. The most common skin problems in DM are acanthosis nigricans, necrobiosis lipoidica, diabetic dermopathy, scleredema, and granuloma anulare. The purpose of this review is to describe the molecular and anatomopathological alterations occurring at the skin during DM, and to illustrate the most important and common clinical skin manifestations in patients with DM. .

The tendon of the long head of the biceps in complex proximal humerus fractures: a histological perspective.

We have studied the histologic and immunohistochemical changes of the long head of the biceps brachii tendon (LHB) in low-energy complex proximal humerus fractures. Our objective was to detect histological features, which may be correlated to pain generation. Biopsy samples were obtained during hemiarthroplasty procedures from 11 patients who suffered a complex proximal humerus fracture. The control group consisted of 10 samples harvested from human cadavers with no history of premortem shoulder problems and no gross shoulder pathology. Histologic investigation included quantitative measurement of tendon degeneration, cellularity, neoangiogenesis, inflammation and metaplasia, as well as immunohistochemical detection of cells with neural differentiation within the tendon tissue proper with S-100 protein and neuropeptide Y (N-Y). The found lesions were significantly more in the group of tendons from fractures compared to the control group (p<0.001). These lesions were also statistically correlated to each other, indicating a possible neural differentiation of tendon stromal cells. The LHB is a potential source of pain and the routine use of tenotomy/tenodesis of this tendon in hemiarthroplasty procedures for fracture may be reinforced by the results of this study.