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Decreased expression of Heat Shock proteins may lead to compromisedwound healing in type 2 diabetes mellitus patients

Kanhaiya Singh, Neeraj K. Agrawal, Sanjeev K. Gupta, Gyanendra Mo-han, Sunanda Chaturvedi, Kiran Singh

PII: S1056-8727(15)00008-2DOI: doi: 10.1016/j.jdiacomp.2015.01.007Reference: JDC 6389

To appear in: Journal of Diabetes and Its Complications

Received date: 1 December 2014Revised date: 9 January 2015Accepted date: 11 January 2015

Please cite this article as: Singh, K., Agrawal, N.K., Gupta, S.K., Mohan, G., Chaturvedi,S. & Singh, K., Decreased expression of Heat Shock proteins may lead to compromisedwound healing in type 2 diabetes mellitus patients, Journal of Diabetes and Its Complica-tions (2015), doi: 10.1016/j.jdiacomp.2015.01.007

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Kanhaiya et al. : Heat shock proteins and impairment of wound healing in T2DM cases

Decreased expression of Heat Shock proteins may lead to compromised wound healing in

type 2 diabetes mellitus patients

Kanhaiya Singh1, Neeraj K Agrawal

2, Sanjeev K Gupta

3, Gyanendra Mohan

4, Sunanda

Chaturvedi4 and Kiran Singh

1*

1Department of Molecular & Human Genetics, Banaras Hindu University,

Varanasi-221005, India

2Department of Endocrinology and Metabolism, Institute of Medical Sciences,

Banaras Hindu University, Varanasi-221005, India

3Department of Surgery, Institute of Medical Sciences, Banaras Hindu University,

Varanasi-221005, India

4Indian Railway Cancer Hospital and Research Centre, N.E.R.,

Varanasi- 221002, India

1* Correspondence

Kiran Singh

Department of Molecular & Human Genetics

Banaras Hindu University

Varanasi-221005, India

e-mail: singhk4@rediffmail.com, skiran@bhu.ac.in

Telefax: +91-542-670-2499

Telephone: +91-9454210058

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Abstract

Background: Heat shock proteins (HSPs) are inducible stress proteins expressed in cells

exposed to stress. HSPs promote wound healing by recruitment of dermal fibroblasts to the site

of injury and bring about protein homeostasis. Diabetic Wounds are hard to heal and inadequate

HSPs may be important contributors in the etiology of diabetic foot ulcers (DFU).

Objective: To analyze the differential expression of HSPs and their downstream molecules in

human diabetic wounds compared to control wounds.

Methods: Expressional levels of HSP27, HSP47 and HSP70 and their downstream molecules

like TLR4, p38-MAPK were seen in biopsies from 101 human diabetic wounds compared to 8

control subjects without diabetes using RT- PCR, western blot and immunohistochemistry.

Results: Our study suggested a significant down regulation of HSP70, HSP47 and HSP27 (p

value = <0.001 for HSP70; p value = 0.007 for HSP47; p value = 0.007 for HSP27) in DFU

along with their downstream molecules TLR4 and p38-MAPK (p value = 0.006 for p38-MAPK;

p value = 0.02 for TLR4). HSP70 levels were significantly lower in male subjects and their

levels increased significantly with the grades of wound on Wagner’s scale. Infection status of the

wounds was found to be significantly associated with the increased levels of HSP70 and HSP27

in infected diabetic wounds.

Conclusions: Our study demonstrates that the down regulation of HSPs in diabetic wounds is

associated with wound healing impairment in T2DM subjects.

KEY WORDS: Heat shock proteins; Wound healing impairment; type 2 diabetes mellitus

(T2DM); TLR4; MAPK; Diabetic foot ulcer

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Introduction

Heat shock proteins (HSPs) are heterogeneous gene products of a highly conserved family of

stress proteins [1]. Their molecular weights vary roughly from 16 KDa to 110 KDa, and they are

rapidly expressed in cells exposed to a variety of stress [2]. All organisms ranging from

archeabacteria to eubacteria, or from plants to animals, respond to endogenous or exogenous

stress by inducing HSPs [1]. The synthesis and expression of these stress proteins is regulated

mainly by a transcription factor known as heat shock factor-1 (HSF-1) which bind to the heat

shock elements (HSE) present in the promoter region of specific genes. In addition to this

emergency response, they also serve as molecular chaperones in various physiological and

pathological conditions by bringing about the folding of nascent polypeptides and targeting

improperly folded proteins for degradation [3]. These proteins maintain a state of homeostasis

during normal cell growth as well as in pathological condition by maintaining the cellular

integrity [4]. Recent reports have suggested that HSPs after their release in the blood, also

participate in signal transduction [5]. These proteins have anti-inflammatory properties as they

inhibit nitric oxide synthase activity along with NF-κB dependent gene expression [6]. Emerging

evidences also suggest certain members of HSPs participate in both innate and adaptive

immunity [7]. HSPs can modulate cellular adaptive response by modulating CD8+ cytotoxic cell

receptor on one hand and can directly stimulate innate immune response by toll like receptor

(TLR) mediated signaling on the other [5, 8].

Wound healing is a dynamic process which includes 4 main overlapping stages ranging from (i)

acute inflammation through (ii) fibrin rich exudates organization, (iii) re-epithelialization to (iv)

granulation tissue formation [9]. The healing wound bed contains different inducible HSPs like

HSP90, HSP70, HSP47 and HSP27 which all together bring about protein homeostasis and cell

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proliferation during wound healing [10]. HSP90 brings about wound healing by supporting the

differentiation of keratinocytes [11]. HSP70 lowers oxidative damage of fibroblasts and supports

cell proliferation in the wound area by inhibiting stress induced apoptosis and TLR activation

[12]. HSP70 is also a ligand for TLR4, which is shown to be an important contributor to wound

closure [13]. The functioning of HSP70 is aided by expression of HSP47, which is

predominantly involved in pro-collagen synthesis and binding with collagen type II and III [14].

Small HSPs like HSP27 also supports wound healing by stabilizing actin microfilaments,

supporting endothelial cell migration in the wound bed, protecting sensory neuron degeneration

and inhibiting stress induced apoptosis [15]. This function of HSP27 is governed by its MAPK

mediated phosphorylation [16].

Type 2 diabetes mellitus (T2DM) is characterized by chronic hyperglycemia and is a proximal

determinant of secondary complications like neuropathy, tissue ischemia and infection [17]. The

wounds in T2DM patients often turn into non healing micro-environment leading to chronic

ulcers due to less oxygen supply, death and deformity in migrating fibroblasts, abnormal matrix

degradation and oxidative stress [18, 19]. T2DM also regulates the HSP synthesis by modulation

of transcriptional or translational processes [20]. Deficient insulin signaling in diabetic

individuals lead to glycogen synthase kinase 3-β (GSK-3β) mediated inhibitory phosphorylation

and deactivation of HSF1.This result in the low levels of intracellular HSPs (iHSPs) especially in

insulin sensitive tissues like muscles and liver which further interfere with insulin signaling via

activation of inflammatory cytokines, c-Jun N-terminal kinase (JNK) and IkappaB kinase (pIKK-

β) in a cyclic manner [4]. This decreased level of iHSPs create an impaired stress response in

diabetic individuals manifested by glycation, oxidation and aggregation of cellular proteins

which in majority of cases lead to the disruption of homeostatic processes[4]. An array of

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literature supports the deficiency of HSPs especially HSP70 or their transcriptional activators in

diabetic wounds in various model systems [21] but little is known about the role of HSPs in

human diabetic wounds. In the present work we have tried to see the expression levels of HSP27,

HSP47 and HSP70 and their downstream molecules like TLR4, p38 mitogen-activated protein

kinase (p38-MAPK) in biopsies from human diabetic wounds in comparison to non diabetic

wounds.

Materials and methods:

Subjects:

This case control study comprised of 109 subjects in which 101 were Diabetic foot ulcer (DFU)

cases and 8 were controls without having T2DM. All DFU patients included were Diabetics who

had non-healing wounds of > 4 weeks duration, thus qualifying as Diabetic wounds. Majority of

the patients had lower extremity wounds 90% of which were located on the foot alone and in the

remaining 10% foot + lower leg were involved. Both the plantar and dorsal aspects of the foot

were involved in the majority of the cases. The samples were collected at the time of their (the

patients) first visit to the Diabetic foot clinic. Samples were taken from the wound margins

during the debridement process and the histological analysis was performed to determine the cell

types (Sup. Figure1). Classification of wounds was made on the basis of the Wagner’s Grading

System [22]. The presence and absence of infection in the wounds were also recorded. Samples

were collected from the OPD clinics and operation theatres of Department of Endocrinology and

Metabolism and Department of Surgery, Institute of Medical Sciences, Banaras Hindu

University, Varanasi, India during the period of July 2010 to December 2013. Tissue samples

were collected in RNAlater solution (P/N AM7020, Ambion, Inc., Austin, TX, USA) and

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phosphate buffer saline (PBS) for RNA and Protein isolation respectively and kept frozen at -

80°C until use. For immunohistochemical staining, samples were collected in Boiun's fixative

solution and kept at room temperature. Patients underwent a standardized clinical and laboratory

evaluation. The T2DM patients having neuropathic, vascular or traumatic ulcers were included in

this study. Screening for neuropathy was done by taking a history of sensory loss and other

symptoms such as a burning sensation or paresthesias. Clinical neurological examination

included the assessment of the vibratory threshold perception using a 128 Hz tuning fork and

assessment of pain and fine touch with a pin and 10g monofilament respectively. The tendon

reflexes and muscle power were measured in patients with sensory neuropathy. Screening for

vascular involvement included a detailed history of vascular insufficiency, clinical examination

for signs of chronic ischemia and assessment of all lower limb pulses. A bed side hand held

Doppler study was carried out in all clinically suspicious cases and ABPI (ankle brachial

pressure index) of < 0.9 was considered indicative of peripheral vascular disease. Age and sex

matched control tissues were obtained by full thickness wound biopsies of post cellulitic chronic

ulcers of the foot and the distal leg. These were non-healing ulcers present for 4 weeks or more

following cellulitis of the lower limb. Each patient’s family history, habits (smoking, alcoholism

etc.), and disease were recorded through a questionnaire (Table 1). The exclusion criteria of the

study included presence of co morbid disorders such as thyroid dysfunction and patients not

belonging to north India. The study was approved by the Institutional Human Ethics Committee

of Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. Informed written

consent was obtained from every participant of each group.

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Semi-quantitative RT-PCR

Total RNA was isolated from wounds samples using TRIzol reagent followed by DNase

treatment. cDNA was synthesized and semi-quantitative RT-PCR analysis of HSP70, HSP47

HSP27, p38-MAPK and TLR4 was done in 101 DFU cases and 8 controls. Sequences of the

implicated primers in the study are provided in Table 2. The PCR conditions were initial

denaturation step of 94°C for 5 min followed by 30 cycles of 30 sec at 94°C, 40 sec at 58°C, 40

sec at 72°C and then a final extension step of 10 min at 72°C. Glyceraldehyde 3-phosphate

dehydrogenase (GAPDH) expression level was checked as an internal control to ascertain the

quality of cDNA. Expression of gene transcripts were quantified after normalizing samples using

GAPDH gene.

Quantitative Real-time PCR

A quantitative RT-PCR (RT-qPCR) experiment was also performed to validate the results

obtained by semi-quantitative RT-PCR in 88 DFU samples of different grades on Wagner Scale

and 8 controls. RT-qPCR experiment was performed according to the manufacturer’s protocol

(Applied Biosystem) using primers for HSP27, HSP47, HSP70 and GAPDH. Briefly, 20 μl total

reaction volume containing 10 μl SYBR Green, 0.1 μl each forward and reverse primer (10pm/

μl) and 2 μl cDNA was used in PCR using ABI 7500 instrument. PCR was performed with an

initial incubation at 50°C for 2 min, then followed by 10 min denaturation at 95°C and 40

cycles at 95°C for 15 s, 60°C for 1 min and 72°C for 15 s. Gene expression profiles were

normalized to the mRNA levels of housekeeping gene GAPDH. ΔΔCT and the relative fold

change of HSPs in DFU cases were calculated according to our previous report [23].

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Immunohistochemical staining

Wound tissue samples obtained during the debridement process were fixed in Bouin’s solution,

embedded in paraffin, and sectioned into 3 µm thick sections. 5 DFU wounds and 5 control

wounds were randomly selected for the study. Anti HSP27 antibody (Catalog No. ab5579,

Abcam Inc., Cambridge, MA., 1:75 in PBS), Anti HSP47 antibody (Catalog No. ab88115,

Abcam Inc., Cambridge, MA., 1:75 in PBS) and Anti HSP70 antibody (Catalog No. ab47455,

clone C92F3A-5, Abcam Inc., Cambridge, MA., 1:75 in PBS) were applied separately to the

deparaffinized sections and incubated in a wet chamber at 4°C for 12 hours. Vectastain Elite

ABC Kit (Vector Laboratories, Burlingame, CA) was used for immunohistochemical staining.

Slides were counterstained with Hematoxylin (Himedia, India). Cells having brown-stained

cytoplasm were regarded as positive. Similar staining time and procedure was adopted for all

tissue samples. Expression patterns of HSP27, HSP47 and HSP70 in control and diabetic wounds

were done under the microscope (Nikon) using different magnifications (4, 10, 20 and 40).

Documentation of acquired images was done using a calibrated digital camera system (Nikon

eclipse 80i) together with the software evaluation package (NIS Elements software). The

expression density HSPs in wound biopsies were computed according to a previous study by

Souil et al. [24].

Western blot

Western blot analysis was performed for HSP27, HSP47 and HSP70 on whole-tissue extracts of

wound biopsies to verify the results of IHC. About 50 μg of protein was loaded on 12 % SDS-

PAGE gel, which was transferred to nitrocellulose membrane and then blocked with 5% of skim

milk in TBS. For HSP27, rabbit polyclonal anti-HSP27 antibody (Catalog No. ab5579, Abcam

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Inc., Cambridge, MA., 1:1000 dilution), for HSP47, mouse polyclonal anti-HSP47 antibody

(Catalog No. ab88115, Abcam Inc., Cambridge, MA., 1:1000 dilution) and for HSP70, mouse

monoclonal anti-HSP70 antibody (Catalog No. ab47455, clone C92F3A-5, Abcam Inc.,

Cambridge, MA., 1:1000 dilution) were used and then incubated with the secondary antibody

linked to horseradish peroxidase. The immunoreactive bands were visualized by the Enhanced

Chemiluminescence System (Amersham Biosciences). Blots were stripped off and reprobed with

anti-GAPDH antibody.

Statistical analysis

The data were expressed as mean considering standard error of mean as error bars. Statistical

significance (P < 0.05) was determined by Student’s t test (two-tailed) and nonparametric

ANOVA. Statistical analysis of data was performed using Graph Pad Prism 5.01 and IBM SPSS

Statistics 20.0 software.

Results

Semi-quantitative RT-PCR analysis showed that there was significant down regulation of

HSP70, HSP47 and HSP27 in wounds of DFU patients compared to control cases (p value =

<0.001, t = 5.59, R squared = 0.22 for HSP70; p value = 0.007, t = 2.77, R squared = 0.06 for

HSP47; p value = 0.007, t = 2.72, R squared = 0.06 for HSP27) (Figure 1, Sup. Figure 2). This

down-regulation of analyzed HSPs message was again confirmed by RT-qPCR analysis (p-value

< 0.001, mean fold change = 2.47 ± 0.15, t = 4.73 for HSP70; p-value < 0.001, mean fold change

= 2.04 ± 0.14, t = 4.17 for HSP47; p-value = 0.03, mean fold change = 2.79 ± 0.15, t = 2.10 for

HSP27) (Figure 1.b). The mRNA level of HSP70 was higher in females as compared to males

(p-value = 0.017, t = 2.42, R squared = 0.06) while the expressions of HSP47 and HSP27

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message were similar in both genders (p-value = 0.63, t = 0.47, R squared = 0.002 for HSP47

and p-value = 0.81, t = 0.23, R squared = 0.001 for HSP27) (Figure 2). HSP70 and HSP27 were

found to be significantly higher in the infected diabetic wounds compared to controls (p-value =

0.012, t = 0.2.55, R squared = 0.06 for HSP70 and p-value = 0.03, t = 2.17, R squared = 0.045

for HSP27) while HSP47 levels remained unaltered even in the presence of infection (p-value =

0.70, t = 0.38, R squared = 0.001 for HSP47) (Figure 3). HSP70 level was found to increased

significantly with the severity of diabetic wounds on Wagner’s scale (p-value = 0.01, t = 3.79, R

squared = 0.105) while other two HSPs levels remained unaltered with the severity of diabetic

wounds (p-value = 0.26, t = 1.35, R squared = 0.041 for HSP47 and p-value = 0.24, t = 1.42, R

squared = 0.04 for HSP27) (Figure 4, Sup. Figure 3). Analysis of mRNA downstream molecules

to HSP27 and HSP70 were also done. Expression of p38-MAPK suggested its similar

expression patterns as HSP27 (Figure 5 a, 5 b, Sup. Figure 4) and TLR4 expression were similar

to the patterns of HSP70 (p value = 0.02, t = 2.30, R squared = 0.05 for TLR4; p value = <0.001,

t = 5.59, R squared = 0.22 for HSP70) (Figure 6 a, 6 b, 6 c, Sup. Figure 5). Western blot analysis

showed that these HSPs were significantly down regulated at translational levels also (p value =

0.001, t = 3.72, R squared = 0.42 for HSP70; p value = <0.0001, t = 5.88, R squared = 0.65 for

HSP47; p value = 0.01, t = 2.77, R squared = 0.31 for HSP27) (Figure 7). Figure 8.A shows the

positive and negative (no primary antibody) staining patterns of different antibodies of HSPs

used in IHC study. Absence of any signal in negative controls suggested that the signal in

positive control was specific signal of primary antibody binding to the antigens.

Immunohistochemical expression analysis between groups also suggested significant down

regulation of HSP70, HSP47 and HSP27 between the wound biopsies of DFU cases and controls

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(Figure 8.B, 8.C, 8.D). The down regulation of TLR4 in DFU cases compared to control wounds

was in a similar pattern to HSP70 (Figure 9, Sup. Figure 6)

Conclusion

The cells of all organisms generally counteract sub lethal endogenous or exogenous stresses by

activating transcription of a specific set of genes termed as HSPs. The stresses may be

physiological (e.g., inflammation, ischemia), pathological (e.g., bacterial or viral infection) or

environmental (e.g. heat shock, oxidative stress or heavy metal poisoning) [25]. Some HSPs are

normally expressed in cells regulated by hormones like estrogens while others are produced

exclusively during stress periods by certain cells [26]. A cell dependent unique pattern of

induction and repression of other genes is followed after expression of HSPs. These HSPs are

highly conserved in amino acid sequences and contain a specific DNA motif called heat shock

element (HSE) [27]. These HSEs when occupied by HSFs bring about the transcription of HSPs.

HSPs are also shown to be protective against insulin resistance and obesity induced

inflammation, thereby abrogating T2DM [28]. Some members of HSPs like HSP90 and HSP70

are also shown to promote wound healing by promoting recruitment of dermal fibroblasts and

endothelial cells to the site of injury, thereby, supporting re-epithelialization in model animals

[29]. Wounds of T2DM patients are hard to heal and inadequate amount of inducible HSPs may

be one of the important contributors in the etiology of DFU [30]. The present work was designed

to correlate the expression levels of three HSPs namely HSP70, HSP47 and HSP27 along with

their downstream interacting partners like p38-MAPK and TLR4 with wound healing

impairment in human T2DM subjects. Several contributing factors like gender of the subjects,

infection status of the wounds and severity of wounds on Wagner’s scale were also taken into

account.

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Immediate release of anti-apoptotic and anti-oxidative HSP70 by keratinocytes prevailing in the

epidermis is a prerequisite of an acute wound [31]. This HSP70 after secretion in the wound

microenvironment bring about the regulation of the inflammatory phase by coordinating pro and

anti-inflammatory responses [32]. Appropriate levels of HSP70 in wounds often lead to abundant

granulation tissue synthesis and proper healing. The analysis of our data indicated that there was

significant down regulation of HSP70 in human diabetic wounds compared to non diabetic

wounds. Our result is supported by the findings of Oberringer et al. (1995), according to which

non-diabetic wounds lacking adequate HSP70 proteins may often develop into chronic decubitus

ulcers [33]. Another report by Vígh et al. (1997) suggested that the application of Bimoclomol, a

strong inducer of HSP70, significantly enhance wound closure in thermally wounded STZ

diabetic rats compared to controls [34]. Another finding that external HSP70 inductions in

chronic wounds of streptozotocin-induced diabetic mice bring about the rapid closure of wounds

also supported our hypothesis that decreased levels of HSP70 is one of the causes of DFU in

humans [35]. Lack of proper activation of innate immunity by TLR4 mediated signaling is also

one of the causes of developing DFU [23, 36, 37]. HSP70 being a ligand for TLR4 is also

required in proper amount in healing wounds [13]. Hence we simultaneously analyzed the

expression levels of HSP70 and TLR4 in DFU subjects and the results obtained indicated that

less expression of HSP70 and TLR4 combined together bring about compromised wound healing

in them. Levels of HSP70 and TLR4 both showed a gender dependency as female subjects had

comparatively higher amount of HSP70 and TLR4 compared to their male partners. Regulation

of HSP70 and TLR4 via estrogen is the reason behind this finding and it supports the higher

incidence of DFU in males compared to females [38, 39]. The levels of HSP70 along with TLR4

were found to be dependent on the grades of wounds and severe wounds were associated with

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higher levels of HSP70 compared to normal wounds. The levels of HSP70 were also found to be

significantly associated with the infection status of the diabetic wounds with higher levels of

HSP70 transcripts in infected diabetic wound compared to non infected wounds. The reason for

this observation may be the finding that microbial infections bring about an increase in

expression of HSP70 in infected cells [40].

HSP27 plays an important role in modulating actin dynamics in response to various stimuli.

HSP27 participates in the wound healing process by regulating fibroblasts and endothelial cell

migration, adhesion and invasion in the wound microenvironment. The expression and spatial

distribution of HSP27 is shown to regulate normal wound healing [41]. This effect of HSP27 is

mainly orchestrated by p38MAPK mediated phosphorylation. HSP27 is only known substrate of

certain mitogen activated protein kinase-activated protein kinase (MAPKAPK) 2/3 [42, 43].

HSP27 is also a protective agent against diabetic neuropathy and hence diminished expression of

HSP27 may also compromise healing in diabetic neuropathic ulcers [44]. The findings of present

work suggested a significant down regulation of HSP27 both at the transcriptional and

translational levels in biopsies of DFU cases compared to controls. The p38-MAPK expression at

transcription levels also followed the similar trend in DFU subjects. This finding of ours was in

coordination with a recent report by Crowe et al. (2013) which suggests that mice deficient in

orthologue of HSP27 display delayed wound healing by lesser collagen deposition and prolonged

inflammatory stage [45]. HSP27 levels like HSP70 was also dependent upon the infection status

of diabetic wounds. This finding is supported by the observation of Wainberg et al. that

cumulative expression of HSP70 and HSP27 by CD4+ lymphocytic cells shoot up following

acute infection of DNA and RNA mediated viruses [46]. The expressional levels of HSP27

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transcripts were found not be associated with grades of wounds and the gender of the DFU

subjects.

HSP47 is also called collagen binding protein 1 and is a molecular chaperone specific for

procollagen (47). Being localized to endoplasmic reticulum, the function of HSP47 is to bring

about the maturation of collagen by mediating post-transcriptional modification in procollagen

[48]. Wang et al. (2002) shown that HSP47 is inducible in wounds and is one of the positive

contributors in healing fetal wounds [49]. Our study showed that HSP47 expression was

significantly lower in diabetic wounds compared to control wounds. This down-regulation of

HSP47 in diabetic wounds may abrogate wound healing by inappropriate collagen synthesis and

less differentiation of fibroblasts into myofibroblasts [50]. This finding of ours is supported by

the report of Wang et al. (2009) which supports the wound healing enhancer capability of HSP47

in alloxan-induced diabetes rats [51]. Unlike HSP70 and HSP27, HSP47 transcripts did not show

any gender, wound grade or wound infection dependency. One limitation of the present study

was the less number of control samples compared to DFU cases, reason being unwillingness of

controls to provide tissue biopsy due to pain and fear associated with the process.

In conclusion, our study demonstrates the combined down regulation of HSP70, HSP27 and

HSP47 expression in diabetic wounds creates a sort of hostile microenvironment in the wound

bed and abrogates wound healing in T2DM subjects by less collagen availability, improper

migration of wound healing fibroblasts, sensory neuropathy promotion and persistent

inflammation. Further research is required to see the effect of activators of HSPs on healing

patterns of diabetic wounds in vitro for effective therapeutic intervention in this respect.

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Conflict of interest

The authors declare no conflict of interest.

Funding

This work was funded by Department of Science and Technology, New Delhi, India (SR/FT/LS-

101/2010). Financial assistance by Department of Biotechnology, Ministry of Science and

Technology, New Delhi, India in form of Senior Research fellowship to the first author is

thankfully acknowledged. We thank Nilu Prasad and Shanti Besra, Laboratory Superintendents,

Indian Railway Cancer Hospital and Research Centre, N.E.R., Varanasi, for their technical

assistance during IHC work.

Author contribution

Kanhaiya Singh designed research, performed experiments, collected and analyzed the data,

wrote the paper. Kiran Singh designed research, interpreted data, and wrote the paper. N.K.A.,

S.K.G., G.M. and S.C. provided samples and did the clinical evaluation of patients. Their critical

comments helped us in writing and approving the final manuscript. Dr. Kiran Singh is the

guarantor of this work, had full access to all the data, and takes full responsibility for the

integrity of data and the accuracy of data analysis.

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Figure Legends:

Figure.1 RT-PCR analysis indicates the decreased expression of HSP70, HSP47and

HSP27 transcripts in DFU patients. RNA was isolated from wound samples of DFU

patients and Controls, cDNA was synthesized and RT-PCR for HSP70, HSP47, HSP27

and GAPDH was performed.

(A) Bar Graph represents the percent ratio which was calculated for the expression of

HSPs and GAPDH in Controls and DFU respectively. Expression of HSP70, HSP47 and

HSP27 transcripts were found to be downregulated in DFU cases as compared to controls

(p value = <0.0001, t = 5.59, R squared = 0.22 for HSP70; p value = 0.007, t = 2.77, R

squared = 0.07 for HSP47; p value = 0.007, t = 2.72, R squared = 0.06 for HSP27.

(B) Bar Graph of qPCR analysis showing the lower expression of HSPs mRNA in the

wounds of DFU patients. Analysis was done in 88 DFU cases and 8 controls. Fold

change in the expression of genes was determined using the ΔΔCT method of relative

quantification. The graph was plotted using log (Relative average fold change) i.e. (log 2-

ΔΔCT). The graph clearly showed that HSP70, HSP47 and HSP27 were down regulated

significantly in the wounds of T2DM cases compared to controls (p-value < 0.001, mean

log (fold change) = - 2.47 ± 0.15, t = 4.73 for HSP70; p-value < 0.001, mean (log fold)

change = - 2.04 ± 0.14, t = 4.17 for HSP47; p-value = 0.03, mean log (fold change) = -

2.79 ± 0.15, t = 2.10 for HSP27). Log (10) = 1; log (100) = 2.

Figure2. Bar Graph showing the correlation of HSP70, 47 and 27 transcripts with

the Gender of DFU patients. Among the diabetic wounds, males showed relatively

lesser amount of HSP70 mRNA transcripts with respect to females (p-value = 0.017, t =

2.42, R squared = 0.06) while the expressions of HSP47 and HSP27 message were

similar in both genders (p-value = 0.63, t = 0.47, R squared = 0.002 for HSP47 and p-

value = 0.81, t = 0.23, R squared = 0.001 for HSP27).

Figure3. Bar Graph showing the correlation of HSP70, 47 and 27 transcripts with

the infection status of DFU wounds. HSP70 and HSP27 were found to be significantly

associated to the infection status of the diabetic wounds. Infected wounds contained

higher levels of HSP70 and HSP27 compared to sterile wounds (p-value = 0.012, t =

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0.2.55, R squared = 0.06 for HSP70 and p-value = 0.03, t = 2.17, R squared = 0.045 for

HSP27). HSP47 levels remained unaltered even in the presence of infection (p-value =

0.70, t = 0.38, R squared = 0.001 for HSP47).

Figure4. Bar Graph showing the comparison of HSP70, 47 and 27 transcripts with

the wound grades on Wagner’s scale. HSP70 message was significantly dependent on

the grade of wounds on Wagner’s scale and its level were high in more severe wounds

compared to less severe wounds (p-value = 0.01, t = 3.79, R squared = 0.105). HSP27

and HSP47 levels remained unaltered with the severity of diabetic wounds (p-value =

0.26, t = 1.35, R squared = 0.041 for HSP47 and p-value = 0.24, t = 1.42, R squared =

0.04 for HSP27).

Figure5. Bar graph showing p38-MAPK having similar expression patterns as

HSP27. (A) Expression of p38-MAPK and HSP27 transcripts were found to be down

regulated in DFU cases as compared to controls (p value = 0.006, t = 2.79, R squared =

0.07 for p38-MAPK; p value = 0.007, t = 2.72, R squared = 0.06 for HSP27). (B) p38-

MAPK message was significantly dependent on the grade of wounds on Wagner’s scale

(p-value = 0.02, t = 3.34, R squared = 0.11) and shared a similar pattern followed by

HSP27 transcripts in DFU wounds.

Figure6. Bar graph showing TLR4 having similar expression patterns as HSP70. (A)

Expression of TLR4 and HSP70 transcripts were found to be significantly down

regulated in DFU cases as compared to controls (p value = 0.02, t = 2.30, R squared =

0.05 for TLR4; p value = <0.001, t = 5.59, R squared = 0.22 for HSP70). (B) Both

HSP70 and TLR4 message were found to be sharing a similar patterns dependent upon

grade of wounds on Wagner’s scale. (C) The mRNA levels of both HSP70 and TLR4

were significantly higher in females as compared to males (p-value = 0.017, t = 2.42, R

squared = 0.06 for HSP70; p value = 0.02, t = 2.3, R squared = 0.06).

Figure7. HSPs protein expression was analyzed in 15 DFU patients and 6 controls.

Tissue samples collected from control {C} and DFU patients {P} were homogenized and

Western blot analysis was performed for expression of HSP70, HSP47, HSP27 and

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GAPDH protein. Bar Graph showing down regulation of HSPs in wounds of DFU

patients compared to controls (p value = 0.001, t = 3.72, R squared = 0.42 for HSP70; p

value = <0.0001, t = 5.88, R squared = 0.65 for HSP47; p value = 0.01, t = 2.77, R

squared = 0.31 for HSP27).

Figure8. Immunohistochemistry of HSP70, 47 and 27 in wound samples with insets

showing detail of staining (40 X magnifications). Microwave-induced antigen retrieval

used 0.01 M citrate buffer (pH 6). Figure 8.A shows the positive and negative (no

primary antibody) staining patterns of different antibodies of HSPs used in IHC study.

Absence of any signal in negative controls suggested that the signal in positive control

was specific signal of primary antibody binding to the antigens. Immunohistochemical

expression analysis among groups also suggested significant down regulation of HSP70,

HSP47 and HSP27 between the wound biopsies of DFU cases and controls (Figure 8.B,

8.C, 8.D).

Figure9. Immunohistochemistry of TLR4 in wound samples with insets showing

detail of staining (40 X magnifications) with Mouse monoclonal anti-TLR4 antibody.

Upper panel shows the negative (no primary) (left) and positive staining (right). Lower

panel shows the Immunohistochemistry for TLR4 in non-diabetic control wound (left)

and diabetic wound (right). TLR4 was found to be down regulated in DFU cases

compared to control wounds in a similar pattern to HSP70.

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Table 1 Biochemical and Demographic parameters of DFU patients (N = 101) and

controls (N = 8). Data are presented as mean ± SD or as number (percentage).

PARAMETERS DFU (N = 101) Control (N = 8) p-value

Age in years; mean ± SD 54.22 ± 8.94 years 56.27 ± 3.42 0.61

BMI in kg/m2; mean ± SD 21.69 ± 2.36 Kg/m2

23.45 ± 1.95 0.15

Duration of T2DM in years; mean ± SD 10.18 ± 4.46 years N/A --

Male 69 (68.32 %) 5 (62.5%) 0.88

Female 32 (31.68 %) 3 (37.5 %) 0.81

HbA1c levels (%) (Mean, range) 10.6 (8.7 to 13) % N/A --

Family history present; n (%) 13 (12.87 %) N/A --

Nephropathy present (Serum creatinine > 1.4 mg/dl); n (%) 30 (29.70%) N/A --

Neuropathy present (by monofilament test); n (%) 61(60.39 %) N/A -

Hypertension present (systolic BP > 140 mm of Hg); n (%) 35(34.65 %) N/A --

Retinopathy present; n (%) 11 (10.89%) N/A --

Dislipidimea present (Serum cholesterol and Tgy levels > 200

mg/dl); n (%)

15(14.85 %) N/A --

Infection present (Wound culture positive for microbes); n (%) 56(55.44 %) N/A --

Bone involvement (Osteomyelitis); n (%) 36 (35.64 %) N/A --

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Table 2: Primers used for RT-PCR analysis of different genes studied and base pair products of

the amplicons.

GENE Forward Primer Reverse Primer Amplicon size

(bp)

HSP70 ACCAAGCAGACGCAGATCTTC CGCCCTCGTACACCTGGAT 73

HSP27 TCCCTGGATGTCAACCACTTCG GGGACAGGGAGGAGGAAACTTG 184

HSP47 CGCCATGTTCTTCAAGCCA CATGAAGCCACGGTTGTCC 70

p38-MAPK ATGCCGAAGATGAACTTTGC TCTTATCTGAGTCCAATACAAGCATC 94

TLR4 CAGAGTTTCCTGCAATGGATCA GCTTATCTGAAGGTGTTGCACAT 85

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