Expression of cell proliferation cycle negative regulators in fibroblasts of an ischemic diabetic foot ulcer. A clinical case report

Abstract

Wound chronification and opportunistic infections stand as major factors leading to lower extremities amputations in diabetes. The molecular mechanisms underlying diabetic's torpid healing have not been elucidated. We present the case of a female diabetic patient that after a plantar abscess surgical drainage, tight glycaemia control and infection clearance; the wound bed evolved to chronification with poor matrix accumulation, scant angiogenesis and no evidence of dermo‐epidermal contours contraction. Ulcer fibroblasts yet cultured under ‘physiological’ conditions exhibited a slow and declining proliferative response. Diabetic fibroblasts cycle arrest occurred earlier than non‐diabetic counterparts. This in vitro premature arrest‐senescence phenotype appeared related to the transcriptional upregulation of p53 and the proto‐oncogene c‐myc; with a concomitant expression reduction of the survival and cellular growth promoters Akt and mTOR. Importantly, immunocytochemistry of the diabetic ulcer‐derived fibroblasts proved nuclear over expression of potent proliferation inhibitors and pro‐senescence proteins as p53 phosphorylated on serine‐15 and p21^Cip(1). In line with this, cyclin D1 appeared substantially underexpressed in these cells. We postulate that the downregulation of the Akt/mTOR/cyclin D1 axis by the proximal activation of p53 and p21 due to stressor factors, impose an arrest/pro‐senescence programme that translated in a torpid and slow healing process.

INTRODUCTION

Wound healing failure is a significant complication of diabetes mellitus associated with increased disability, morbidity and mortality 1, 2, 3, 4. Hyperglycaemia and its associated biochemical disturbances are toxic for granulation tissue cells, acting as deterrents of cutaneous repair. Peripheral artery disease (PAD) is an additional negative ingredient to wound bed cells further restricting wound healing. Here we describe molecular evidences emerged from primarily cultured and processed diabetic wound fibroblasts, which indicate the rupture of physiological axes of cell growth and proliferation that underlie the clinical refractoriness for healing.

CASE PRESENTATION

The case refers to a 67‐year‐old female patient, with a 30‐year history of type 1 diabetes, hypertension and previous diagnosis of bilateral femoro‐popliteal occlusion with no criteria of revascularisation or endoarterial intervention. Distal pulses were absent and the ABI of both limbs did not exceed 0·4. Microvascular disease had been diagnosed and subsequently confirmed by histopathological analysis. She was admitted at the Diabetic Foot Wound Ward of the Institute of Angiology and Vascular Surgery due to a plantar abscess on the anterior portion of the left foot, resulting from a local puncture trauma. At the clinical examination, local signs of acute and severe inflammation were present along with a collection of purulent material. Adjacent tissues appeared necrotic. She was begun on a course of intravenous rocephin, amikacin and metronidazole. The abscess was drained approximately 24 h later and the necrotic material extensively debrided. The local infectious signs appeared controlled 72 h after the intervention. The patient systemic condition gradually improved. In addition, her glucose (4·3–6·2 mmol/l) and haemoglobin (13·4 g/l) remained controlled. Although infection was circumscribed to soft plantar tissues and despite all the pharmacological and medical care provided, the wound evolved slowly. The internal contours were frequently reanimated with debridement until a scant bleeding appeared. A plantar crater of approximately 5·5 cm in diameter and 1·5–2 cm depth remained on postoperative day 12 with slow granulation response and no evidence of contraction or re‐epithelialisation. The wound appeared clinically clean, supported by microbiology laboratory data. Once the slow‐progressing granulation had filled the crater, the patient was discharged on postoperative day 71 after surgical drainage. Re‐epithelialisation was followed under ambulatory regimen within four months from the initial admission.

Samples collection

On day 21, a 3‐mm (diameter) granulation tissue cylinder biopsy was taken from a relatively granulated edge of the crater. The material collected was used for histopathological analysis, and to examine the transcriptional expression (mRNA) and as mature protein of critical targets by immunocytochemistry using low passages (4–5) cultured fibroblasts. Cells were grown under standard culture conditions in DMEM (glucose 5·5 mM) supplemented with 10% foetal calf serum and 95% oxygen. Fibroblast proliferation rate was calculated as described (5) using trypan blue exclusion and haemocytometer manual counting. Oligonucleotides were synthesised for analysing the transcriptional expression of the following genes: p53 (NM_000321.2), c‐myc (NM_002467.4), Akt (NM_005163.2) and mTOR (NM_004958.3) via RT‐PCR. mRNA values were normalised against constitutive expression of RNAr 18 S gene. Immunocytochemistry allowed examining the expression and cellular compartmentalisation of p53 (phosphorylated on serine 15), p21, cyclin D1 and Akt (phosphorylated on serine 473). Each reaction was conducted using as a concurrent control, primarily cultured fibroblasts derived from the granulation tissue of a burn injury of a healthy non‐diabetic adult subject. An anti‐mouse/anti‐rabbit HRP/DAB detection kit was used according to manufacturer's instructions (all reagents were manufactured by Abcam, Cambridge, MA).

The histopathological analysis of the ulcer biopsy confirmed that the granulation tissue derived from ischaemic patients is characterised by (i) limited angiogenesis, (ii) abnormal thickening of neovessels' walls, (iii) fibrohyaline matrix suggestive of ‘hardened’ appearance and (iv) infiltration of immune‐inflammatory cells (lymphocytes and macrophages) (6).

Despite the standard culture conditions, the fibroblasts collected from the ulcer bed grew more slowly and ended growing to a lower confluence than the control counterparts, derived from the granulation tissue of the burn injury (Figure 1). The fibroblasts gene transcriptional characterisation by RT‐PCR including p53, c‐myc, Akt and mTOR (Figure 2), showed a transcriptional increase of 36% for p53 relative to non‐diabetic control cells (0%). The renowned cell proliferation promoter c‐myc, exhibited a 163% transcriptional increase relative to control fibroblasts. Akt and mTOR expression were found reduced to 71 and 89%, respectively, as compared to non‐diabetic healthy donor cells. Immunocytochemistry experiments revealed a robust nuclear expression of p53 with the posttranslational modification of phosphorylation at serine 15. It proved to be massively expressed by the diabetic ulcer‐derived fibroblasts (Figure 3A). In sharp contrast, burn injury‐derived fibroblasts showed no p53 expression under the present experimental conditions (Figure 3B). Concomitantly, the cell‐cycle inhibitor p21^Cip1 was found intensely expressed by the diabetic fibroblasts (Figure 3 C). No nuclear or cytoplasmic p21 expression was detected in the fibroblasts collected from the non‐diabetic burn donor (Figure 3D). Furthermore, the proliferation trigger cyclin D1 appeared underexpressed in the nucleus of the diabetic ulcer fibroblasts (Figure 3E), as opposed to the non‐diabetic counterpart. For the later, a massive and clear immunolabelling was found in both nuclear and cytoplasmatic compartments (Figure 3F). As a corollary, the diabetic ulcer fibroblasts had no expression of an active form of Akt, phosphorylated on serine 473 (Figure 3G), which contrasted with the nuclear and cytoplasmic expression by the non‐diabetic fibroblasts (Figure 3H).

Figure 1.

Time course proliferative response of diabetic and non‐diabetic fibrolasts. Healthy donor (HD) and diabetic fibroblasts (DF) were cultured under physiological glucose (5.5 mM) and oxygen (95%) concentrations supplemented with 10% foetal calf serum. Normal wound fibroblasts exhibit a progressive proliferation rate reaching a plateau after 20 days of culture. Conversly, diabetic ulcer‐derived cells exhibit an irreversible decline after day 10 of culture.

Figure 2.

Gene expression profile by diabetic and non‐diabetic wound fibroblasts. HD, healthy donor; DF, diabetic fibroblasts.

Figure 3.

Immunocytochemistry experiment. (A) Nuclear and cytoplasmic expression of p53 phosphorylated on serine 15 in fibroblasts derived from the diabetic ulcer. (B) Non‐diabetic donor fibroblasts exhibiting no p53 expression. (C) Nuclear and cytoplasmic expression of p21 in fibroplast derived from the diabetic ulcer. (D) Fibroplasts derived from the granulation tissue of an age‐matched non‐diabetic adult with a burn injury showing no expression of p21. (E) Cyclin D1 is not expressed by the fibroplasts derived from the torpidly progressing diabetic ulcer. (F) Clear nuclear and cytoplasmic expression of cyclin D1 in the fibroplasts derived from the burn injury of a non‐diabetic subject. (G) Fibroplasts derived from the ischemic diabetic ulcer do not show expression of the active Akt. (H) Akt phosphorylated in ser 473 is expressed by the healing burn injury fibroplasts. Expression involves the membrane, cytoplasm and nucleus.

DISCUSSION

Diabetes impairs most if not all the events encompassed within the cutaneous healing cascade. The fibroblast is an instrumental cell for the healing process by secreting, contracting and remodelling the scar, as by contributing to re‐epithelialisation. Interestingly, this patient's fibroblasts exhibited the typical proliferative impairment of chronic wounds' cells (7), even when cultured under standard conditions of normoxia and glucose and serum concentrations. Thus, this supports the notion that diabetic wound fibroblasts exhibit a sort of ‘behavioural memory’. Although high glucose burden and hypoxia are stressor factors that dismantle wound fibroblasts and endothelial cells physiology; the molecular mechanisms underlying the diabetes‐associated fibroblasts' proliferation reluctance and premature senescence have not been enlightened. However, the identification of a relative transcriptional increase of p53 and c‐myc contribute to explain such phenotypes. p53 is largely known as a cell‐cycle checkpoint that can arrest from G₁/S up to G₂/M. Although the finding on the c‐myc overexpression may appear contradictory; our observation confirms that c‐myc overexpression represents one of the many p53 collaborative loops for arresting cells (8) and that c‐myc supraphysiological levels lead to proliferative arrest (9). Furthermore, it is clinically meaningful the transcriptional reduction detected for Akt and mTOR as both are proximal transducers of survival and cells growth signals. mTOR for instance, responds to contextual thresholds of oxygen (hypoxia) and substrates limitation via the IGF‐1/Akt pathway (10).

Immunocytochemistry using phosphorylation targeted antibodies rendered further molecular explanation for the clinical slow healing. Significantly, the nuclear expression of p53 with the posttranslational modification of phosphorylation at serine 15, massively detected in the diabetic ulcer‐derived fibroblasts, has been considered as an amplification of the p53 ability to induce cell‐cycle arrest (11). Although this modification can be experimentally induced by a variety of stressors (12), including glucose availability in vitro (13); its detection in cells from a torpidly progressing ischaemic ulcer seems pathophysiologically meaningful. Furthermore, the robust and exclusive expression of p21^Cip1 in the diabetic fibroblasts nucleus is also significant and consequential. This protein is identified as a p53 downstream effector arm, acting as a cyclin‐dependent kinase inhibitor promoting cell‐cycle arrest (14). Overexpression of p21^Cip1 in venous ulcers fibroblasts has been associated with the onset of a non‐proliferative, and senesce programme (15). In close biological correspondence to the previous observations, cyclin D1 appeared substantially underexpressed in the nucleus of the diabetic ulcer fibroblasts. Cyclin D1 is responsible for quiescent cells to enter and progress throughout the cell cycle (16). In pressure ulcers for instance, the cyclin D/cdk4 complex enabled to estimate the proportion of the fibroblast population that is capable of contributing to wound repair, thus acting as healing predictor (17). Finally, we detected that the diabetic ulcer fibroblasts had no expression of the active form of Akt, phosphorylated on serine 473. Akt functional depletion via mTOR downregulation or by hypoxia may imply that these diabetic fibroblasts are more vulnerable to apoptosis, proliferative arrest and to onset autophagy (18).

Interpretively, our findings suggest that this hard‐to‐heal phenotype is preceded by the downregulation of proliferative and pro‐anabolic pathways as a result of the existence of one or multiple local upstream triggers as ischaemia, inflammation or reactive radicals, that may activate p53 (via its phosphorylation on serine 15) as major cellular homeostatic monitor. In response, activated p53 activates p21, which are able to negatively regulate the Akt/mTOR axis, and proliferative (mdm2, cyclin D) pathways that abort cell division. To our knowledge, this is the first description in which coherently converge different molecular clues that explain the bases of clinical recalcitrance and chronification of a diabetic ulcer.

ACKNOWLEDGEMENTS

The authors declare no actual or potential conflicts of interest with respect to the authorship and/or publication of this article.